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WORKS  OF 
PROFESSOR    J.    A.    MANDEL 

PUBLISHED   BY 

JOHN  WILEY  &  SONS. 


Handbook   for  Biochemical  Laboratory. 

12mo,  cloth,  $1,50. 

TRANSLATIONS. 

A  Text=bool4  of  Physiologrical  Ctiemistry. 

By  Olof  Hammarsten,  late  Professor  of  Medical  and 
Physiological  Chemistry  in  th3  University  of  Upsala 
Authorized  Iranslafon,  from  the  author's  ei-larged 
and  revised  Gth  Ge  man  ed  t  on,  by  John  A.  Man- 
del,  Professor  of  Chemisty  iu  the  New  York  Univer- 
sity and  Bc-Uevue  Hospital  Medical  College.  8vo, 
viii  +  845  pages,  cloth,  $4  OU. 

A  Compendium  of  Chemistry,  Including:  General,   In- 
org:anic,  and  Orsranic  Chemistry. 

By  Dr  Carl  Ari  old  Professor  of  C  hem  stry  in  the 
Royal  Veterinary  School  of  Hannover.  Au  horized 
translation  from  the  eleventh  enlirged  and  revised 
German  edition,  by  John  A  Mandel.  Small  8vo, 
xii  +  6_'7  pages,  cloih,  $3.50. 


A  TEXT-BOOK 


PHYSIOLOGICAL  CHEMISTRY. 


OLOF  HAMMARSTE:^^, 

Late  Professor  of  Medical  and  Physiological  Chemistry  in  the 
University  of  Upsala . 


FMOM  THE  AUTHOR'S  ENLARGED  AND  REVISED 
SIXTH  GERMAN  EDITION 


JOHN  A.   MANDEL,   Sc.D., 

Professor  of  Chemistry  in  the  Xew  York  University  and 
Bellevue  Hospital  Medical  College. 


FIFTH     EDITION, 
FIRST    THOUSAND. 


NEW   YORK: 

JOHN  WILEY  &  SONS. 

LoxDOx:    GHAPMAK^  &  HALL,  LnriTED. 
1908. 


Copyright,  1893,  1898,  1900,  1904,  1908, 

BY 

JOHN  A.  MANDEL. 

a  "F  ^'1  "^ 


Sl)e  Sri fttllfir  ^rpBa 
i&abrrt  Qrummanb  anh  Qlomtiang 


TRANSLATOR'S   PREFACE   TO   THE   FIFTH    AMERICAN 

EDITION. 


Since  the  appearance  of  the  First  American  Edition  of  Hammarsten's 
Physiologischen  Chemie  in  1893  it  has  become  more  and  more  popular  as 
a  text-book  in  our  medical  schools,  and  has  acquired  much  favor  among 
the  biochemical  research  workers  of  this  country. 

A  special  debt  of  gratitude  is  owing  to  Professor  Hammarsten  for  his 
many  revisions,  which  have  enabled  this  work  to  keep  jjace  with  all  the 
advances  of  this  department  of  chemical  science.  The  same  sound 
critical  survey  of  the  subject  characterizes  this  as  well  as  the  past 
editions. 

I  am  under  obligations  to  Dr.  Charles  B.  Robinson  for  much  assistance 
in  proof  revision. 

John  A.  Ma.ndel. 

New  York,  December,  1907. 

V 


PREFACE  TO    THE  SIXTH  GERMAN    EDITION. 


Although  only  a  short  time  has  elapsed  since  the  appearance  of  the 
Fifth  Edition,  the  enormous  advances  in  the  different  domains  of  physio- 
logical chemistry  have  necessitated  a  thorough  revision  of  each  of  the 
chapters  and  a  rewriting  of  most  of  them.  B3cause  of  these  facts  it  has 
been  impossible  to  prevent  an  increase  in  the  size  of  the  book,  and  hence 
this  edition  is  somewhat  larger  than  the  preceding  ones.  Acceding  to  the 
wishes  expressed  by  many  friends,  an  Authors  Index  has  been  added  to 
this  edition,  but  in  other  respects  the  plan  of  the  work  has  not  been 
changed. 

Olof  Hammarsten. 

IJpsAiA,  September  8,  1906. 


CONTENTS. 


CHAPTER  I. 

PAGE 

I STPODUCTION 1 

CHAPTER  II. 
The  Protein  Substances 26 

CHAPTER  III. 
The  Carbohydrates 104 

CHAPTER  IV. 
The  Animal  Fats 131 

CHAPTER  V. 
The  Animal  Cell 139 

CHAPTER  VI. 
The  Blood 170 

CHAPTER  VII. 
Chyle,  Lymph,  Transudates,  and  Exudates 250 

CHAPTER  VIII. 
The  Liver 280 

CHAPTER  IX. 
Digestion 339 

CHAPTER  X. 

Tissues  of  the  Connective  Substance 428 

vii 


VlU  CONTENTS. 

CHAPTER  XI. 

PAGE 

Muscles 447 

CHAPTER  XII. 
Brain  and  Nerves 479 

CHAPTER  XIII. 
Organs  of  Generation 495 

CHAPTER  XIV. 
Phe  Milk 514 

CHAPTER  XV. 
The  Urine 541 

CHAPTER  XVI. 
The  Skin  and  its  Secretions 685 

CHAPTER  XVII. 
Chemistry  of  Respiration 696 

CHAPTER  XVIII. 
Metabolism 715 

General  Index 779 

Index  to  Authors 813 


PHYSIOLOGICAL   CHEMISTRY. 


CHAPTER  I. 
INTRODUCTION. 

It  follows  from  the  law  of  the  conservation  of  matter  and  of  energy  that 
living  beings,  plants  and  animals,  can  produce  neither  new  matter  nor  new 
energy.  They  are  only  called  upon  to  appropriate  and  assimilate  already 
existing  material  and  to  transform  it  into  new  forms  of  energy. 

Out  of  a  few  relatively  simple  combinations,  especially  carbon  dioxide 
and  water,  together  with  ammonium  compounds  or  nitrates,  and  a  few 
mineral  substances,  which  serve  as  its  food,  the  plant  builds  up  the  extremely 
complicated  constituents  of  its  organism,  proteins,  carbohydrates,  fats, 
resins,  organic  acids,  etc.  The  chemical  work  w^hich  is  performed  in  the 
plant  must  therefore,  in  the  majority  of  cases,  consist  in  syntheses;  but 
besides  these,  processes  of  reduction  take  place  to  a  great  extent.  The 
radiant  energy  of  the  sunlight  induces  the  green  parts  of  the  plant  to  split 
off  oxygen  from  the  carbon  dioxide  and  water,  and  this  reduction  is  generally 
considered  as  the  starting-point  of  the  following  syntheses.  According 
to  a  hypothesis  suggested  by  A.  Baeyer,i  at  first  formaldehyde  is  pro- 
duced, C02  +  H20  =  CH20  +  02,  which  by  condensation  is  transformed  into 
sugar,  and  this  then  serves  in  the  structure  of  other  bodies.  The  energy 
of  the  sun,  which  produces  this  splitting,  is  not  lost;  it  is  only  transformed 
and  is  stored  as  chemical  energy  in  the  new  compounds  produced  in  the 
synthesis.  W.  Loeb^  has  been  able  to  obtain  formaldehyde  as  a  direct 
reaction  product  from  CO2  and  H2O  by  the  aid  of  the  silent  electric  dis- 
charge.    Th6  formation  of  aldehyde  takes  place  in  the  three  following 


*  Ber.  d.  d.  chem.  Gesellsch.,  3.  '  Zeitschr.  f.  Elektrochem.,  12. 


2  INTRODUCTION. 

phases:  first,  2C02  =  2CO  +  02;  second,  CO+H20  =  C02+H2;  and  third, 
C0+H2  =  HC0H.  The  formation  of  sugar  from  CO2  and  H2O  with  the 
introduction  of  energy  can  be  expressed  by  the  following: 

1.  C02+H20  =  c6  +  H2  +  02. 

2.  H2+C0  =  HC0H. 

3.  2(H2  +  CO)  =  CH20H.CHO. 

4.  6HCOH  =  C6Hi206. 

5.  3CH20H.CHO  =  CoHi206. 

In  animal  life  the  conditions  are  not  the  same.  Animals  are  dependent 
either  directly,  as  the  herbivora,  or  indirectly,  as  the  carnivora,  upon  plant- 
life,  from  which  they  derive  the  three  chief  groups  of  organic  nutritive, 
matter — proteins,  carbohydrates,  and  fats.  These  bodies,  of  which  the 
protein  substances  and  fats  form  the  chief  mass  of  the  animal  body,  undergo 
within  the  animal  organism  a  cleavage  and  oxidation,  and  yield  as  final 
products  exactly  the  above-mentioned  chief  components  in  the  nutrition  of 
plants,  namely,  carbon  dioxide,  water,  and  ammonia  derivatives,  which  are 
rich  in  Oxygen  and  have  little  energy.  The  chemical  energy,  which  is 
partly  represented  by  the  free  oxygen  and  partly  stored  up  in  the  above- 
mentioned  more  complex  chemical  compounds,  is  transformed  into  other 
forms  of  energy,  principally  heat  and  mechanical  work.  While  in  the  plant 
we  find  chiefly  reduction  processes  and  syntheses,  which  by  the  introduc- 
tion of  energy  from  without  produce  complex  compounds  having  a  greater 
content  of  energy,  we  find  in  the  animal  body  the  reverse  of  this,  namely, 
cleavage  and  oxidation  processes,  which,  as  we  used  to  state,  convert 
chemical  tension  into  living  force. 

This  difference  between  animals  and  plants  must  not  be  overrated,  nor 
must  we  consider  that  there  exists  a  sharp  boundary-line  between  the  two. 
This  is  not  the  case.  There  are  not  only  lower  plants,  free  from  chloro- 
phyll, which  in  regard  to  chemical  processes  represent  intermediate  steps 
between  higher  plants  and  animals,  but  the  difference  existing  between  the 
higher  plants  and  animals  is  more  of  a  quantitative  than  of  a  qualitative  kind. 
Plants  require  oxygen  as  peremptorily  as  do  animals.  Like  the  animal,  the 
plant  also,  in  the  dark  and  by  means  of  those  parts  which  are  free  from 
chlorophyll,  takes  up  oxygen  and  eliminates  carbon  dioxide,  while  in  the 
light  the  oxidation  processes  going  on  in  the  green  parts  are  overshadowed 
or  hidden  beneath  the  more  intense  reduction  jirocesses.  As  in  the  animal, 
we  also  find  a  heat  production  in  fermentation  produced  by  plant  organisms; 
and  even  in  a  few  of  the  higher  plants — as  the  aroideoe  when  bearing  fruit — 
a  considerable  development  of  heat  has  been  observed.  On  the  other 
hand,  in  the  animal  organism,  besides  oxidation  and  splitting,  reduction 
processes  and  syntheses  also  takes  place.  The  contrast  which  seemingly 
exists  between  animals  and  plants  consists  merely  in  that  in  the  animal 
organism  the  processes  of  oxidation  and  splitting  are  predominant,  while 


ANIMAL  OXIDATIONS.  3 

in  the  plant  chiefly  those  of  reduction  and  synthesis  have  thus  far  Ijeen 
studied. 

WoHLER  1  in  1824  was  the  first  to  observe  an  example  of  synthetical 
PROCESSES  within  the  animal  organism.  He  showed  that  when  benzoic  acid 
is  introduced  into  the  stomach  it  reappears  as  hippuric  acid  in  the  urine, 
after  combining  with  glycocoU  (aminoacetic  acid).  Since  the  discovery  of 
this  synthesis,  which  may  be  expressed  by  the  following  equation: 

CeHs.COOH  +  NH2.CH2.COOH  =  NH  (C6H5.CO).CH2.COOH  +H2O, 

Benzoic  acid  GlycocoU  Hippuric  acid 

and  which  is  ordinarily  considered  as  a  type  of  an  entire  series  of  syntheses 
occurring  in  the  body  where  water  is  eliminated,  the  number  of  known 
syntheses  in  the  animal  kingdom  has  increased  considerably.  ]\Iany  of 
these  syntheses  have  also  been  artificially  produced  outside  of  the  organism, 
and  numerous  examples  of  animal  syntheses  of  which  the  course  is  abso- 
lutely clear  will  be  found  in  the  following  pages.  Besides  these  well-studied 
syntheses,  there  occur  in  the  animal  body  also  similar  processes  unquestion- 
ably of  the  greatest  importance  to  animal  life,  but  of  which  we  know 
nothing  with  positiveness.  We  enumerate  as  examples  of  this  kind  of 
synthesis  the  re-formation  of  the  red-blood  pigment  (the  haemoglobin),  the 
formation  of  the  different  proteins  from  simpler  substances,  and  the  produc- 
tion of  fat  from  carbohydrates.  This  last-mentioned  process,  the  formation 
of  fat  from  carbohydrates,  is  also  an  example  of  reduction  processes  which 
occur  to  a  considerable  extent  in  the  animal  body. 

Formerly  the  view  was  generally  accepted  that  animal  oxidation  takes 
place  in  the  fluids,  while  to-day  we  are  of  the  opinion,  derived  from  the 
investigations  of  Pfluger  and  his  pupils,^  that  it  is  connected  with  the 
form-elements  and  the  tissues.  The  question  as  to  how  this  oxidation  in 
the  form-elements  is  induced  and  how  it  proceeds  cannot  be  answered  with 
certainty. 

When  a  substance  is  oxidized  by  neutral  oxygen  at  the  ordinar\'  tempera- 
ture or  at  the  temperature  of  the  body,  the  substance  is  said  to  be  easily  oxidized 
or  autooxidized,  and  the  process  is  considered  as  a  direct  oxidation  or  auto- 
oxidation.  As  the  oxygen  of  the  inspired  air,  and  that  of  the  blood,  is  neutral 
molecular  oxygen,  the  old  assumption  that  ozone  occurs  in  the  organism  has 
now  been  discarded  for  several  reasons.  On  the  other  hand,  the  chief  groups 
of  organic  nutritives,  carbohydrates,  fat,  and  proteins,  the  last  two  forming 
•  the  chief  mass  of  the  animal  body,  are  not  autooxidizable  substances.  They 
are   on   the   contrary  bradoxidizable    (Traube)    or   dysoxidizable  bodies. 


'  Berzelius,  Lehrb.  d.  Chemie,  ubersetzt  von  Wohler,  4,  p.  356,  Abt.  1,  Dresden,  1831. 
^  Pfluger,  Pfliiger's  Archiv.  6  and  10;    Finkler,  ibid.,  10  and   14;    Oertmann,  itid.t 
14  and  15;  Hoppe-Seyler,  ibid.,  7. 


4  INTRODUCTION. 

They  are  nearly  indifferent  to  neutral  oxygen,  and  it  is  therefore  a  question 
how  an  oxidation  of  these  and  other  dysoxidizable  bodies  is  possible  in  the 
animal  bod}'. 

In  explanation  it  is  very  generally  admitted  that  the  oxygen  is  made 
active  and  this  causes  a  secondary  oxidation.  It  is  generally  conceded  that 
in  autooxidation  a  cleavage  of  neutral  oxygen  takes  place.  The  autooxidiz- 
able  substance  splits  the  oxygen  molecule  and  combines  with  one  of  the 
oxygen  atoms,  while  the  other  free  atom  as  active  oxygen  may  oxidize  the 
dysoxidizable  substances  simultaneously  present.  Such  a  subordinate 
oxidation  is  called  an  indirect  or  secondary  oxidation.  The  explanation 
of  animal  oxidations  has  been  attempted  in  different  ways  by  the  sup- 
position that  the  oxygen  is  made  active  and  thus  produces  secondary 
oxidation. 

The  cause  of  the  animal  oxidation  is  considered,  by  Pfltjger  and 
several  other  investigators,  to  be  dependent  upon  the  special  constitution  of 
the  protoplasmic  proteins  or  the  living  protoplasmic  substance.  This 
investigator  calls  the  proteins  outside  of  the  organism,  or  those  which 
occur  in  the  animal  fluids,  "non-li\dng  proteins,"  and  considers  them  to  be 
somewhat  different  from  those  occurring  in  living  protoplasm.  The  latter 
are  called  "living  proteins"  (PFLiJGER),  "active  proteins  "  (Loew),  or  "bio- 
gens"  (Verworn).  The  living  protoplasmic  molecule  differs  from  the 
ordinary  non-living  protein  b}^  being  more  unstable  and  therefore  having  a 
greater  inclination  towards  intramolecular  changes  of  the  atoms.  The 
reason  for  these  greater  intramolecular  movements  Pfluger  ascribes  to  the 
presence  of  cyanogen,  and  Latham  attributes  it  to  the  presence  of  a  chain 
of  cyanalcohols  in  the  protein  molecule.  Verworn,i  on  the  contrary,  claims 
an  intramolecular  introduction  of  oxygen  into  a  large  hypothetical  proto- 
plasmic molecule,  the  "biogen  molecule,"  which  is  supposed  to  contain  a 
nitrogen  or  an  iron  complex  as  an  oxygen  receptor  or  carrier,  and  a  side- 
chain  of  aldehydic  character  like  that  of  the  carbohydrates,  as  an  oxidizable 
group. 

According  to  Loew,^  who  bases  his  claim  upon  special  investigations 
and  numerous  toxicological  observations,  the  unstability  of  the  active 
proteid  molecule  is  due  to  the  simultaneous  presence  of  aldehyde  and 
unstable  amino  groups.  These  occur  separated  from  each  other  in  the 
active  proteins,  and  when  they  combine  the  protoplasm  dies,  the  molecule 
being  changed  into  a  stable  condition,  i.e.,  into  dead  protein.  It  is  also 
a  fact  that  all  substances  which  react  with  aldehyde  and  unstable  amino 
groups  are  poisonous  to  the  living  cells. 

*  Pfliigcr,  Pfliiger's  Archiv,  10;  Latham,  Brit.  Med.  Journal,  1886;  Verworn, 
Die  Biogenhypothese,  Jena,  1903. 

'  Loew  and  Bokomy,  Pfliiger's  Archiv,  25;  0.  Loew,  ibid.,  30;  and  specially 
0.  Loew,  The  Energy  of  Living  Protoplasm,  London,  1896. 


ANIMAL   OXIDATIONS.  5 

LoEW  has  also  shown,  in  conjunction  with  Bokorxy,  that  in  many 
plants  a  verj'  unstable  reserve-protein  substance  occurs,  which  to  a  cer- 
tain extent  occupies  an  intermediate  position  between  protein  and  organizec 
living  substance. 

The  explanation  as  to  the  oxidation  process  differs  entirely  accord- 
ing to  the  conception  of  the  structure  of  the  unstable  protoplasmic  mole- 
cule. If  the  living  protoplasmic  protein  is  not,  like  protein  in  the  ordinary 
sense,  indifferent  to  neutral  oxygen,  we  can  admit  of  a  cleavage  of  the 
oxygen  molecule  by  this  change.  The  protein  would  be  itself  oxidized, 
while  on  the  other  hand  a  secondary  oxidation  of  other  difficultly  oxidiza- 
ble  substances  could  be  brought  about  by  the  oxygen  atoms  set  free. 

Another  very  wideh'  difTused  view  exists  in  regard  to  the  origin  of  the 
activity  of  the  oxygen,  namely,  that  by  the  decomposition  processes  in  the 
tissues,  reducing  substances  are  formed  which  split  the  neutral  oxygen 
molecule,  miiting  with  one  oxygen  atom  and  setting  the  other  free. 

The  formation  of  reducing  substances  during  fermentation  and  putre- 
faction is  generally  known.  The  butyric  fermentation  of  dextrose  in  which 
hydrogen  is  set  free — C6Hi206  =  C4H802-f 2C02-r2H2 — is  an  example  of 
this  kind.  Another  example  is  the  appearance  of  nitrates  in  consequence 
of  an  oxidation  of  nitrogen  in  cases  of  putrefaction,  which  process  is  ordi- 
narily explained  by  the  statement  that  reducing,  easily  oxidizable  bodies 
are  formed  which  split  oxygen  molecules,  liberating  oxygen  atoms  which 
aftenvard  oxidize  the  nitrogen.  It  is  assumed  also  that  the  cells  of  the 
animal  tissues  and  organs  have  the  power,  like  these  lower  organisms 
which  produce  fermentation  and  putrefaction,  of  causing  splitting  processes 
in  which  easily  oxidizable  substances,  perhaps  also  nascent  hydrogen 
(Hoppe-Seyler  1),  are  produced. 

In  accordance  with  what  has  been  stated  above  on  the  oxidations  of 
the  animal  body,  primarily  a  cleavage  of  the  organic  constituents  of  the 
body  takes  place  with  the  formation  of  readily  oxidizable  substances. 
The  oxidation  of  these  latter  produces  an  activation  of  the  oxygen  and 
hence  may  also  cause  a  secondary  oxidation  of  dysoxidizable  substances. 
The  products  formed  by  these  splittings  and  oxidations  may  perhaps  in 
part  be  burned  within  the  body  without  undergoing  further  cleavage,  but 
more  probably  they  must  first  undergo  a  further  cleavage  and  then  succumb 
to  consecutive  oxidations,  until  after  repeated  cleavages  and  oxidations 
the  final  products  of  metabolism  are  formed. 

An  activation  of  the  oxygen  may  be  produced  according  to  0.  Nasse^ 
by  a  hydroxylization  of  the  constituents  of  the  protoplasm  with  the  split- 
ting off  of  molecules  of  water.     If  benzaldehvde  is  shaken  with  water  and 


'  Pflliger's  Archiv,  12. 

-  0.  Xasse,  Rostocker  Zeitung,  No.  534,  1S91,  and  No.  363,  lS9o, 


6  INTRODUCTION. 

air,  an  oxidation  of  the  benzaldehyde  into  benzoic  acid  takes  place,  while 
oxidizable  substances  present  at  the  same  time  may  also  be  oxidized. 
The  simultaneous  presence  of  potassium  iodide  and  starch  or  tincture  of 
guaiacum  causes  a  blue  coloration  because  the  hydroxyl  (OH)  takes  the 
place  of  the  hydrogen  in  the  aldehyde  group,  and  these  two  hydrogen 
atoms,  one  derived  from  the  aldehyde  and  the  other  from  the  water,  have 
a  splitting  action  on  the  molecular  oxygen.  Nasse  and  Rosing  ^  have  also 
found  that  certain  varieties  of  protein  have  the  property  of  being  hydroxy  1- 
ized  in  the  presence  of  water.  According  to  Nasse  a  whole  series  of  oxida- 
tions in  the  animal  body  may  be  accounted  for  by  the  oxygen  atoms  set 
free  in  hydroxylization  similar  to  that  of  benzaldehyde.  In  opposition  to 
this  view  we  must  remark  that  the  oxidation  of  benzaldehyde  to  benzoic 
acid  may  also  take  place  in  other  ways,  thus  by  the  intermediary  formation 
of  a  peroxide  (see  Baeyer  and  Villiger;  Engler  and  Weissberg^). 

By  quantitative  methods  van't  Hoff  and  his  pupils  ^  have  shown 
that  molecular  oxygen  can  be  divided  in  two  parts  by  certain  autooxida- 
tion  processes.  One  of  these  unites  wuth  the  autooxidizer  and  the  other 
with  a  body  simultaneously  present  but  not  directly  oxidizable,  which,  ac- 
cording to  the  suggestion  of  Engler,^  is  called  the  acceptor,  a^an't  Hoff 
claims  that  the  oxygen  molecule  dissociates  at  ordinary  temperatures  into 
minimum  quantities  of  positively  and  negatively  charged  oxygen  atoms, 
the  ions  of  similar  charge  uniting  with  the  autooxidizable  substance, 
while  the  remaining  ions  oxidize  the  acceptor.  Such  a  division  of  the 
oxygen  into  two  halves  has  also  been  shown  by  other  investigators,  such 
as  Maxchot,  Engler,  and  his  collaborators.^  These  investigators  never- 
theless consider  that  autooxidation  takes  place  in  another  way,  namely,  by 
the  formation  first  of  peroxides  by  the  taking  up  of  oxygen  molecules. 

Traube  ®  has  also  expressed  a  similar  view.  According  to  him,  in 
autooxidation  we  have  to  deal,  in  the  first  place,  not  with  a  cleavage  of  the 
oxygen,  but  with  a  splitting  of  water  in  which  the  hydroxyl  groups  of  the 
water  combine  with  the  oxidizable  substance,  while  the  hydrogen  atoms 
set  free  on  the  decomposition  of  the  water  unite  with  the  neutral  oxygen, 
forming  hydrogen  peroxide,  which  may  naturally  also  have  an  oxidizing 
action. 

A+2H20  +  02  =  A(OH)2  +  H202. 

'  E.  Rosing,  Untersuchungen  liber  die  Oxydation  von  Eiweiss  in  Gegenwart  von 
Schwefel.     Inaug.  Dissert.     Rostock,  1891. 

'  Baeyer  and  Villiger,  Ber.  d.  d.  chem.  Gesellsch. ,  33;  Engler  and  Weissberg,  ibid.,  33. 

^  van't  Hoff,  Zeitschr.  f.  physikal.  Chem.,  16;  Jorissen,  Ber.  d,  d.  chem.  Gesellsch., 
30,  and  Zeitschr.  f.  physikal.  Chem.,  22;  Ewan,  ibid.,  16. 

■*  Ber.  d.  d.  chem.  Gesellsch.,  33. 

'  Manchot,  tJber  freiwillige  Oxydation,  Leipzig,  1900;  Engler  and  Weissberg, 
Ber.  d.  d.  chem.  Gesellsch.,  33;   Engler  and  Frankenstein,  ibid.,  34. 

'  Ber.  d.  d.  chem.  Gesellsch.,  15,  18,  19,  22,  and  26. 


ANIMAL   OXIDATIONS.  7 

According  to  the  view  of  Engler  and  his  collaborators,  which  corre- 
sponds in  great  measure  with  those  of  Bach  and  of  Manchot,^  at  least  in 
the  simplest  cases  ("direct  autooxidation  "  according  to  Engler),  the 
•oxygen  molecules  unite  with  the  activating  body  (A),  forming  a  peroxide- 
like substance  which  can  give  up  one  of  the  two  oxygen  atoms  to  an 
acceptor  (B): 

A  +  02  =  A02     and     A02  +  B  =  AO  +  BO. 

If  this  is  so,  still  we  do  not  know  to  what  extent  such  peroxides  are 
formed  in  the  oxidation  in  the  living  cell.  The  possibility  of  a  production 
of  peroxides,  and  also  of  hydrogen  peroxide,  in  animal  oxidation  is  still 
generally  admitted,  andCnoDAT  and  Bach  ^  have  indeed  been  able  to  show 
a  peroxide  formation  in  plants.  Still,  if  hydrogen  peroxide  were  formed 
in  such  oxidations  it  would  have  no  further  physiological  importance, 
according  to  Loew,  because  the  animal  and  plant  cells  contain  special 
enzymes,  called  by  him  catalases,  which  quickly  decompose  the  hydrogen 
peroxide  with  the  production  of  molecular  oxygen.  According  to  Loew^ 
the  physiological  importance  of  the  catalases  is  to  protect  the  cell  from 
hydrogen  peroxide,  which  acts  as  a  protoplasmic  poison. 

Loew,'*  who  has  also  opposed  the  view  as  to  the  oxygen  becoming  active 
with  the  setting  free  of  oxygen  atoms,  has  sought  for  the  reason  of  the 
oxidations  in  the  unstable  properties  of  the  protoplasmic  proteins.  The 
active  movement  of  the  atoms  within  the  active  protein  molecule  is  trans- 
mitted to  the  oxygen  and  to  the  oxidizable  substance,  and  when  the  dis- 
solution of  the  molecule  has  proceeded  to  a  certain  point  the  oxidation 
occurs  by  virtue  of  the  chemical  affinity.  The  reason  for  this  unstable 
condition  of  living  protein  molecules  has  already  been  given  above. 

Schmiedeberg,^  who  also  denies  the  supposition  that  the  oxygen 
becomes  active,  is  of  the  view  that  the  tissues  by  the  mediation  of  the 
oxidations  do  not  increase  the  oxidizing  activity  of  the  oxygen,  but  more 
probably  act  on  the  oxidizing  substances,  making  them  more  susceptible 
to  oxidation. 

All  the  views  presented  thus  far  assume  a  continuous  oxidation  of  the 
primary  active  substance.  The  view  has  also  been  suggested  that  animal 
oxidation  may  be  brought  about  by  oxygen-carriers,  i.e.,  by  bodies  which, 


'  Engler  and  Wild,  Ber.  d.  d.  chem.  Gesellsch.,  30;  Bach,  Le  Moniteur  scientifique, 
1897,  and  Compt.  rend.,  124;   Manchot,  1.  c. 

^  Ber.  d.  d.  chem.  Gesellsch.,  3o  u.  36. 

'  Loew,  U.  S.  Dept.  of  Agriculture,  Rep.  68,  1901,  and  Ber.  d.  d.  chem.  Gesellsch., 
35;  in  regard  to  the  opposed  views  see  Chodat  and  Bach,  1.  c,  and  Kastle  and  Loeven- 
hart,  Amer.  Chem.  Joum.,  29. 

*0.  Loew,  The  Energy  of  Living  Protoplasm,  London,  1896. 

*Arch.  f.  exp.  Path.  u.  Pharm.,  1-t. 


8  INTRODUCTION. 

according  to  the  older  views,  without  being  oxidized  themselves,  act  in  an 
analogous  manner  to  the  nitric  oxide  in  the  manufacture  of  sulphuric  acid 
by  alternately  taking  up  and  giving  off  oxygen  in  the  oxidation  of  dys- 
oxidizable  bodies.  Traube  has  for  a  long  time  explained  the  oxidations 
of  the  animal  body  in  this  way,  and  he  calls  these  c^uestionable  oxygen- 
carriers  oxidation  ferments} 

It  has  also  been  positively  proved  by  the  researches  of  Jaquet,  Sal- 
KOWSKi,  Spitzer,  Rohmann,  Abelous  and  Biarnes,  Bertrand,  Bour- 
QUELOT,  De  Rey-Pailhade,  Medwedew,  Pohl,  Jacoby,  Chodat  and  Bach.^ 
and  others  that  in  the  blood  and  different  tissues  of  the  animal  body,  as  also 
in  plant-cells,  substances  occur  which  have  the  property  of  causing  certain 
oxidations  and  are  therefore  called  oxidation  ferments  or  oxidases.  The 
nature  and  mode  of  action  of  these  bodies  will  be  discussed  elsewhere  in 
this  volume,  hence  it  will  be  sufficient  here  to  state  that  in  general  two 
different  groups  of  oxidation  ferments  are  recognized.  The  ferments  of  the 
first  group,  called  primary  or  direct  oxidases  or  simply  oxidases,  transfer 
the  oxygen  of  the  air  directly  to  other  bodies.  Those  of  the  second  group, 
the  indirect  oxidases  or  peroxidases,  are  active  only  in  the  presence  of  a 
peroxide,  as  they  set  oxygen  free  from  these  latter  by  decomposition. 

The  many  different  views  in  regard  to  the  oxidation  processes  show 
us  strikingly  how  little  is  positively  known  about  these  processes.  There 
is  no  doubt  that  the  animal  body  possesses  in  the  so-called  oxidation  fer- 
ments important  means  of  bringing  about  oxidative  decomposition  of  various 
substances,  and  the  occurrence  of  numerous  intermediary  metabolic  prod- 
ucts in  the  animal  body  teaches  us  that  the  oxidation  of  the  constituents 
of  the  body  is  not  instantaneous  and  sudden,  but  takes  place  step  by  step, 
and  hand  in  hand  with  cleavages.  Most  investigators  are  agreed  that 
these  decompositions  are  similar  to  certain  oxidations  studied  by  Drechsel  ^ 
outside  the  animal  body,  where  oxidations  and  reductions  alternate  in  quick 
succession.  The  views  are  divided  in  regard  to  the  manner  and  origin  of 
this  cooperative  action.* 

The  oxidations  in  the  animal  body  have  long  been  designated  as  a 

'  M.  Traube,  Theorie  der  Ferment wirkungen,  Berlin,  1858. 

^Jaquet,  Arch.  f.  exp.  Path.  u.  Pharm.,  29;  Salkowski,  Centralbl.  f.  d.  med.  Wis- 
sensch.,  1892  and  1894,  and  Virchow's  Arch.,  1-47;  Spitzer,  Pfliiger's  Archiv,  60  and 
07;  Spitzer  and  Rohmann,  Ber.  d.  deutsch.  chem.  Gesellsch.,  28;  Abelous  et  Biarnes, 
Arch,  de  physiol.  (5),  7,  8,  and  9,  and  Compt.  rend.  Soc.  biol.,  46;  Bertrand,  Arch,  de 
physiol.  (5),  8,  9,  and  Compt.  rend.,  122,  123,  124;  Bourquelot,  Compt.  rend.  Soc. 
biol.,  48,  and  Compt.  rend.,  123;  Jacoby,  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt. 
1,  which  contains  the  literature  of  the  subject;  Chodat  and  Bach,  1.  c, 

3  Joum.  f.  prakt.  Chem.  (N.  F.),  22,  29,  38,  and  Festschrift  fiir  C.  Ludwig,  1887. 

■*  See  M.  Nenckl  Arch,  des  sciences  biol.  de  St,  P6tersbourg,  1,  483;  Abelous  and 
Aloy,  Compt.  rend.,  136,  137;  Kastle  and  Elvove,  Amer,  Chem.  Joum.,  31;  Underhill 
and  Closson,  Amer.  Joum.  of  Physiol.,  13. 


CLEAVAGES  IX  THE  ANIMAL  BODY.  9 

combustion,  and  such  a  conception  is  easily  reconcilable  with  the  above- 
mentioned  views.  In  combustion  in  the  ordinary  sense,  as,  for  example, 
the  burning  of  wood  or  oil,  we  must  not  forget  that  the  substances  them- 
selves do  not  com]:)ine  with  oxygen.  It  is  only  after  the  action  of  heat 
has  decomposed  these  bodies  to  a  certain  degree  that  the  oxidation  of  the 
products  of  such  decomposition  takes  place  and  is  accompanied  by  the 
phenomenon  of  light. 

The  essential  source  of  heat  and  mechanical  work  developed  in  the 
organism  is  to  be  found  in  the  oxidations.  Chemical  energy  is  transformed 
into  the  above-mentioned  forms  of  energy  in  cleavage  processes,  where 
complicated  chemical  compounds  are  reduced  to  simpler  ones,  and  there- 
fore the  atoms  change  from  an  imstable  to  a  stabler  equilibrium,  and 
stronger  chemical  affinities  are  satisfied.  The  animal  body  may  also  have 
a  source  of  energy  in  the  cleavage  processes  which  are  not  dependent  on 
the  presence  of  free  oxygen.  The  processes  taking  place  in  the  living 
muscle  are  an  example  of  this  kind.  A  removed  muscle,  which  gives  off 
no  oxA'gen  when  in  a  vacuum,  may,  as  Hermann  ^  has  shown,  work,  at  least 
for  a  time,  in  an  atmosphere  devoid  of  oxygen,  and  give  off  carbon  dioxide 
at  the  same  time. 

Cleavage  processes  which  are  accompanied  by  a  decomposition  of  water 
and  then  a  taking  up  of  its  constituents  are  called  hydrolytic  cleavages. 
These  cleavages,  which  play  an  important  role  within  the  animal  body, 
and  which  are  most  frequently  met  with  in  the  processes  of  digestion,  are 
exemplified  by  the  transformation  of  starch  into  sugar  and  the  splitting 
of  neutral  fats  into  the  corresponding  fatty  acids  and  glycerine: 

C3H5(Ci8H350o)3  +  3H20  =  C3H5(OH)3  +  3(Ci8H3602). 
Tristearin  Glycerine  Stearic  acid 

As  a  rule  the  hydrolytic  cleavage  processes  as  they  occur  in  the  animal 
body  may  be  performed  outside  of  it  by  means  of  higher  temperatures 
with  or  without  the  simultaneous  action  of  acids  or  alkalies.  Considering 
the  two  above-mentioned  examples,  we  know  that  starch  is  converted  into 
sugar  when  it  is  boiled  with  dilute  acids,  and  also  that  the  fats  are  split 
into  fatty  acids  and  glycerine  on  heating  them  with  caustic  alkalies  or  by 
the  action  of  superheated  steam.  The  heat  or  the  chemical  reagents  which 
are  used  for  the  performance  of  these  reactions  would  cause  immediate 
death  if  applied  to  the  living  body.  Consequently  the  animal  organism 
must  have  other  means  at  its  disposal  which  act  similarh',  but  in  such  a 
manner  that  they  may  work  without  endangering  the  life  or  normal  con- 
stitution of  the  tissues.  Such  means  have  been  recognized  in  the  so-called 
unorganized  ferments  or  enzymes. 

Untersuch.  iiber  den  Stoffwechsel  der  Menschen,  Berlin,  1867. 


10  INTRODUCTION. 

Alcoholic  fermentation  and  other  processes  of  fermentation  and  putre^ 
faction  are  dependent  upon  the  presence  of  living  organisms,  ferment 
fmigi,  and  splitting  fungi  of  different  kinds.  The  ordinary  view,  according 
to  the  researches  of  Pasteur,  is  that  these  processes  are  to  be  considered  as 
phases  of  the  life  of  these  organisms.  The  name  organized  ferments  or  fer- 
ments has  been  given  to  such  micro-organisms,  of  which  ordinary  yeast  is  an 
example.  However,  the  same  name  has  also  been  given  to  certain  bodies 
or  mixtures  of  bodies  of  unknown  organic  origin  which  are  products  of  the 
<:•  hemic al  work  within  the  cell,  and  which  after  they  are  removed  from  the 
t'ell  still  have  their  characteristic  action.  Such  bodies — for  example,  malt 
diastase,  rennin,  and  the  digestive  ferments — are  capable  in  the  very  small- 
est quantity  of  causing  a  decomposition  or  cleavage  in  very  considerable 
quantities  of  other  substances,  without  entering  into  permanent  chemical 
combination  with  the  decomposed  body  or  with  any  of  the  cleavage  or 
decomposition  products.  These  formless  or  unorganized  ferments  are 
generally  called  enzymes,  according  to  KtJHXE. 

A  ferment  in  a  more  restricted  sense  is  therefore  a  living  being,  while 
an  enzyme  is  a  product  of  chemical  processes  in  the  cell,  a  product  which 
has  an  individuality  even  without  the  cell,  and  which  may  be  active  when 
^separated  from  the  cell.  The  splitting  of  invert-sugar  into  carbon  dioxide 
and  alcohol  by  fermentation  is  a  fermentative  process  closely  connected 
with  the  life  of  the  yeast.  The  inversion  of  cane-sugar  is,  on  the  contrary, 
an  enzymotic  process  caused  by  one  of  the  bodies  or  a  mixture  of  bodies 
formed  by  the  living  ferment,  which  can  be  severed  from  this  ferment,  and 
still  remain  active  even  after  the  death  of  the  latter.  Consequently  fer- 
ments and  enzymes  are  capable  of  manifesting  a  different  behavior  towards 
<'ertain  chemical  reagents.  Thus  there  exist  a  number  of  substances, 
among  which  we  may  mention  arsenious  acid,  phenol,  toluene,  salicylic 
acid,  boracic  acid,  sodium  fluoride,  chloroform,  ether,  and  protoplasmic 
poisons,  which  in  certain  concentration  kill  ferments,  but  which  do  not 
noticeably  impair  the  action  of  the  enzymes. 

The  above  view  as  to  the  difference  between  ferments  and  enzymes  has 
lately  been  essentially  shaken  by  the  researches  of  E.  Buchner  i  and  his 
pupils.  He  has  been  able  to  obtain  from  beer-yeast,  by  grinding  and 
strong  pressure,  a  cell  fluid  rich  in  protein  which  when  introduced  into 

'  E.  Buchner,  Ber.  d.  devitsch.  chem.  Gesellsch.,  30  and  31;  E.  Buchner  and  Rapp, 
xbid.,  31,  32,  34;  H.  Buchner,  Sitzungsber.  d.  Gesellsch.  f.  Morphol.  u.  Physiol,  in 
Miinchen,  13,  1897,  part  1,  which  also  contains  the  discussion  on  this  topic.  See  also 
E.  and  H.  Buchner  and  M.  Hahn,  Die  Zymasegarung,  Miinchen,  1903;  Stavenhagen, 
Ber.  d.  deutsch.  chem.  Gesellsch.,  30;  Albert  and  Buchner,  ibid.,  33;  Buchner,  ibid., 
33;  Albert,  ihid.,  33;  Albert,  Buchner,  and  Rapp,  ibid.,  35;  in  regard  to  the  opposed 
views  see  Macfadyen,  Morris,  and  Rowland,  ibid.,  33;  Wroblewski,  Centralbl.  f. 
Physiol,,  13,  and  Joum.  f.  prakt.  Chem.  (N.  F.),  64. 


FERMENTS  AND  ENZYMES.  11 

a  solution  of  a  fermentable  sugar  caused  a  violent  fermentation.  The 
objections  raised  from  several  sides  that  the  fluid  expressed  still  contained 
dissolved  living  cell  substance  has  been  so  successfully  answered  by  Buch- 
NER  and  his  collaborators  that  there  is  at  present  no  question  but  that 
alcoholic  fermentation  is  caused  by  a  special  enzyme  called  zymase  which 
is  formed  in  the  yeast-cell. 

As  from  the  yeast-cell  so  also  from  other  lower  organisms,  indeed  from 
the  lactic-acid  bacilli  and  beer-vinegar  bacteria,  we  have  recently  been  able 
to  isolate  enzymes  (E.  Buchner  and  Meisexheimer,  Herzog  i)  which 
produce  the  specific  fermentative  action  of  the  mother  organism.  The 
question  whether  there  exist  ferment  processes  which,  in  Pasteur's  sense, 
are  the  result  of  the  biological  phenomena  connected  with  the  metabolism 
of  the  micro-organism  and  which  we  can  directly  identify  with  the  life 
processes,  is  very  difficult  to  answer;  hence  for  the  present  we  have  no 
foundation  for  a  sharp  differentiation  between  the  organized  ferments  and 
enzymes.  The  metabolic  processes  of  the  living  organisms  which  we 
recognize  as  fermentation  phenomena  must  as  a  rule  be  ascribed  to  enzymes 
acting  within  the  cell.  If  such  processes  are  closely  connected  with  the 
life  of  the  cell,  then  this  is  explained  in  part  by  the  fact  that  this  special 
enzyme  is  produced  only  by  living  cells  and  in  part  by  the  fact  that  it 
cannot  be  separated  from  the  living  cells  or  that  it  is  readily  destroyed  on 
the  death  of  the  cell. 

All  enzymes  are  organic  substances  formed  in  the  cells,  whose  chemical 
nature  has  unfortunately  not  been  established  at  the  present  time.  Thus 
far  no  enzyme  has  been  prepared  in  a  pure  state  with  positiveness.  The 
enzymes  are  considered  as  protein  bodies  by  many  investigators,  but  this 
opinion  has  not  sufficient  foundation,  and  is  disputed  at  least  for  certain 
enzymes.  It  is  indeed  true  that  the  enzymes  isolated  by  certain  investi- 
gators acted  like  genuine  protein  bodies;  but  it  is  uncertain  whether  or  not 
the  products  isolated  in  these  instances  were  pure  enzymes  or  were  com- 
posed of  enzymes  contaminated  with  proteins. 

The  enzymes  may  be  extracted  from  the  cells  and  tissues  by  means  of 
water  or  glycerine,  especially  by  the  latter,  which  forms  very  stable  solu- 
tions and  hence  is  extensively  used  as  a  means  of  extracting  them.  The 
enzymes,  generally  speaking,  do  not  appear  to  be  diffusible,  and  Bredig  ^ 
has  given  several  reasons,  which  will  be  given  later,  for  considering  them 
not  as  true  solutions  but  rather  colloidal  ones.  The  enzymes  are  also 
absorbed  by  other  colloids  and  are  carried  down  by  fine  precipitates,  and 
this  property   is  extensively   taken   advantage  of   in  their  preparation.^ 

'  E.  Buchner  and  J.  Meisenheimer,  Ber.  d.  d.  chem.  Gesellsch.,  36;  Herzog,  Zeitschr. 
f.  physol.  Chem.,  37. 

^  Anorganische   Fermente,   Leipzig,   1901. 
'  See  Briicke,  Wien.  Sitzungsber.,  43,  1861, 


12  INTRODUCTION. 

The  manner  of  combination  of  the  enzymes  with  the  colloids  has  not  been 
explained  and  is  no  doubt  not  the  same  in  all  cases.^  They  are  precipitated 
from  their  solutions  by  alcohol.  All  enzymes  lose  their  specific  action  on 
Ijoiling  their  aqueous  solutions,  and  this  is  generally  considered  as  an  im- 
portant criterion  as  to  the  ferment  nature  of  a  body.  The  continued  heating 
of  their  solutions  above  80°  C.  generally  destroys  the  enzymes.  In"  the 
dry  state,  however,  certain  enzymes  may  be  heated  to  100°  or  indeed 
to  150-160°  C.  without  losing  their  activity.  Light  can  also  destroy  en- 
zymes in  watery  solution,  as  shown  wuth  malt  diastase  (Emmerling)  and 
chymosin  (Emmerling,  Schmidt-Nielsen).- 

The  action  of  the  enzymes  may  be  markedly  influenced  by  external  con- 
ditions. The  reaction  of  the  liquid  is  of  special  importance.  Certain 
enzymes  act  only  in  acid;  others,  and  the  majority,  on  the  contrary,  act 
only  in  neutral  or  alkaline  liquids.  Certain  of  them  act  in  very  faintly 
acid  as  well  as  in  neutral  or  alkaline  solutions,  but  best  at  a  specific  reac- 
tion. They  are  all  destroyed  by  concentrated  mineral  acids  and  alkalies. 
The  temperature  exercises  also  a  very  important  influence.  In  general 
the  activity  of  enzymes  increases  to  a  certain  limit  with  the  temperature. 
This  optimum  is  not  always  the  same,  but,  as  shown  by  Tammann,  depends, 
like  the  destructive  action  of  high  temperatures,  essentially  upon  the 
quantity  of  enzyme  and  other  conditions.  The  products  of  the  enzymotic 
processes  exercise  a  retarding  influence  in  proportion  as  they  accumulate, 
and  indeed  the  enzymotic  process  may  thereby  be  entirely  stopped.  In 
such  cases  of  "false  equilibrium"  (Bredig)  we  may,  as  shown  by  Tam- 
mann,^ often  start  the  reaction  again  by  removing  the  products  of  the  re- 
action, by  diluting  with  water,  by  raising  the  temperature,  by  the  addition 
of  more  substance,  or  by  the  addition  of  more  of  the  enzyme.  The  addition 
of  neutral  salts  and  other  substances  of  various  kinds  has  in  some  cases 
an  accelerating,  and  in  other  cases  a  retarding  action.^ 

The  velocity  of  the  enz3'me  action  and  the  final  condition  at  the  conclu- 
sion of  the  enzymotic  processes  is  not  only  dependent  upon  the  reaction, 
the  temperature,  and  the  presence  of  transformation  products  or  of  foreign 
bodies,  but  also  upon  the  amount  of  enzyme  present  and  the  concentration 
of  the  solution.  The  velocity  increases  regularly  with  an  increase  in  tlie 
amount  of  enzyme,  but  not  in  the  same  proportion  with  all  enzymes,  as  it 
has  been  shown  for  different  enz3'mes  that  they  require  different  times  for 

'  Dauwe,  Hofmeister's  Beitrage,  6. 

^  Emmerling,  Ber.  d.  d.  chem.  Gesellsch.,  34;  Schmidt-Nielsen,  Hofmeister's  Bei- 
trage, o. 

^  The  work  of  Tammann  may  l)e  found  in  Zeitschr.  f.  physiol.  Chem.,  16,  and 
Zeitschr.  f.  physikal.  Chem.,  3  and  IS. 

*  See  Fermi  and  Pernossi,  Zeitschr.  f.  Hygiene,  18;  also  in  regard  to  the  enzymes 
in  general  see  C.  Oppenheimer,  Die  Fermente,  2.  Aiid..  IJOo. 


ENZYMES.  13 

action.  This  will  be  discussed  later.  The  concentration  of  the  solution 
is  also  of  great  importance,  and  the  result  of  a  change  of  this  durino-  enzy- 
motic  action  is  of  special  importance  in  the  study  of  the  kinetics  of  enzyme 
reactions. 

We  have  no  characteristic  reactions  for  all  enzymes  in  general,  but 
each  enzyme  is  characterized  by  its  specific  action  and  by  the  conditions 
under  which  it  operates.  Of  special  importance  is,  first,  the  fact  that  the 
enzymes  do  not  form  permanent  chemical  combinations  in  definite  pro- 
portions by  weight  with  the  bodies  upon  which  they  act,  or  their  decompo- 
sition products;  and,  secondly,  that  an  insignificantly  small  amount  of 
the  enzyme  can  decompose  a  relatively  enormous  amount  of  substance. 
For  instance,  1  part  of  invertin  can  invert  100,000  parts  of  cane-sugar 
(O'SuLLivAN  and  Thompson  ^),  and  1  part  of  chymosin  can  in  a  short  time 
decompose  more  than  400,000  parts  of  casein  (Hammarsten^).  This  does 
not  exclude  the  possibility  of  a  primary,  but  temporary,  combination  of  the 
enzymes  with  the  substances  acted  upon.  Such  an  assumption  is,  indeed, 
substantiated  by  the  work  of  Hanriot,  Henri,  Armstrong,^  and  others, 
while,  according  to  Oppenheimer,*  we  can  represent  ferment  action  as 
consisting  of  a  first  phase  where  combination  of  the  enzyme  and  the  sub- 
stance occurs,  and  a  second  phase  where  after  this  combination  a  chemical 
decomposition  of  the  substance  occurs  according  to  the  laws  of  catalysis. 
This  view  coincides  best  with  the  specificity  of  enzyme  action. 

The  specific  action  of  the  enzymes  is  of  special  importance,  as  one  and 
the  same  enzyme  acts  only  upon  one  substance  or  a  definite  group  of  sub- 
stances. Their  action  seems  to  be  entirely  dependent  upon  the  stereo- 
metric construction  of  the  substance  acted  upon,  and  we  may  assume 
that  the  enzyme  attacks  only  specially  arranged  stereometric  atomic 
groups,  where  the  enzyme  fits  the  substance  in  a  manner  similar  to  a  key 
fitting  a  lock  (E.  Fischer).  E,  Fischer  ^  has  given  a  positive  proof  for 
the  great  importance  of  a  different  stereometric  configuration  by  his  inves- 
tigations upon  the  artificially  prepared  series  of  stereoisomeric  glucosides 
which  he  calls  a-  and  /9-glucosides.  The  enzymes  of  yeast  infusions  act 
only  upon  the  glucosides  of  the  a-series,  while  emulsin,  on  the  contrarj', 
acts  only  upon  those  of  the  /3-series. 

Of  especially  great  importance  for  a  deeper  insight  into  the  manner 
of  enzyme  action,  we  must  mention  the  investigations  which  have  been 


'  O'SuUivan  and  Thompson,  Joum.  of  Chem,  Soc,  57. 
'  See  Maly's  Jahresbericht,  7. 

'  Hanriot,  Compt.  rend.,  132;   Henri,  Lois  generates  de  Taction  des  diastases,  Paris, 
1903,  and  Arch,  di  Fisiol.,  1  and  2;   Armstrong,  Proc.  Roy.  Soc.  London,  73. 
*Die  Fermente,  2.  Aufl.,  1903,  p.  66. 
"Zeitschr.  f.  physioL  Chem.,  26. 


14  INTRODUCTION. 

carried  on  recently  on  the  relationship  of  inorganic  catalyzers  to  the 
enzymes,  which  have  thrown  light  upon  the  correspondence  between 
catalysis  and  enzyme  action.  The  catalyzers,  like  the  enzymes  or  their 
derivatives,  are  not  found  in  the  final  products  of  the  reaction,  they  are  not 
used  up  in  the  process,  and  the  quantity  of  the  active  substance  propor- 
tionate to  the  quantity  of  substance  transformed  is  infinitesimally  small 
in  enzyme  action  as  well  as  in  catalysis.  In  both,  the  reaction  velocity 
also  seems  to  be  independent  of  the  quantity  of  the  active  substance  added, 
and  this  indicates  that  the  enzyme  action  is  not  to  be  considered  as  the 
starting  of  a  reaction  which  would  not  of  itself  take  place,  but  rather  as 
an  acceleration  of  a  slowly  proceeding,  often  not  noticeable,  chemical 
change.  According  to  this  conception  enzyme  action  comes  in  line  with 
catalysis,  for,  according  to  Ostwald,i  bodies  are  called  catalyzers  which 
b}'  their  presence  cause  a  change  in  the  reaction  velocity  of  chemical  proc- 
esses, and  indeed  positive  or  negative,  according  as  they  produce  accelera- 
tion or  retardation.  The  striking  correspondence  between  enzymes  and 
inorganic  catalyzers  has  been  shown  especially  by  Bredig  and  his  collaljo- 
rators,  v.  Bernek,  Ikeda,  and  Reinders,^  by  their  very  important  in- 
vestigations. 

Bredig  has  been  able  to  prepare  colloidal  solutions  of  platinum,  gold, 
and  silver  by  allowing  the  electric  arc  to  play  between  two  poles  of  the 
respective  metal  beneath  water.  These  solutions  of  colloidal  metals, 
metallic  sols,  show  in  their  activity  and  the  dependence  of  this  activity 
u})on  external  influences,  and  especially  in  their  destruction  by  poisons, 
such  strong  resemblance  to  the  enzymes  that  Bredig  has  indeed  called  them 
inorganic  ferments. 

Still  it  is  nevertheless  true  that  the  manner  of  action  of  catalyzers 
has  not  been  explained,  and  we  must  be  careful  not  to  draw  too  positive 
conclusions  from  the  remarkable  correspondence  of  the  manner  of  action 
of  metallic  sols  and  certain  ferments.  In  studying  the  action  of  enzymes 
one  is  repeatedly  struck  with  the  marked  deviation  from  the  laws  of  reac- 
tion underlying  inorganic  catalyzers,^  and  this  has  called  forth  a  series 
of  hypotheses  and  attempts  at  explanation,  which  on  account  of  space 
cannot  be  entered  into,  but  we  must  refer  the  reader  to  special  works  on 
the  subject.  On  the  other  hand,  we  must  not  forget  that  the  enzymes  are 
not  pure  substances,  but  are  habitually  mixtures  whose  action  may  be 


'  Gruiidriss  d.  allgemoin.  Chcmie,  3.  Aufl.,  1899. 

*  See  Bredig,  Anorganische  Fermente,  Leipzig,  1901,  and  also  Die  Elemente  d.. 
chemischen  Kinetik,  etc.,  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1,  1902. 

3 See  Brown  and  Glendinning,  Proc.  Chem.  Soc.  18,  1902;  Tammann,  Zeitsehr.  f. 
physikal.  Chem.,  3  .~.i.^  18,  and  Zeitsehr.  f.  physiol.  Chem.,  16;  Henri,  Zeitsehr.  f. 
physikal,  Chem.,  31),  and  Lois  g6n6rales,  etc.  See  also  the  work  of  H,  Euler,  Zeitsehr. 
f.  physiol.  Chem. ,  45. 


ENZYME  ACTIONS.  "  15 

modified  by  an  apparently  insignificant  admixture,  and  for  this  reason 
the  study  of  the  mode  of  action  is  made  very  difficult.  Although  the 
question  as  to  whether  enzymes  follow  the  same  laws  as  the  inorganic 
catalyzers  is  still  an  open  one,  nevertheless  we  know  that  in  a  great 
many  regards  the  enzymes  correspond  with  catalyzers.  The  comparison 
of  these  two  has  opened  up  in  the  study  of  enzyme  action  new  points 
of  elucidation  and  attack  which  have  been  very  fruitful  in  result,  and 
which  have  no  doubt  helped  very  much  in  the  explanation  of  these  difficult 
questions. 

It  is  not  within  the  scope  of  this  book  to  enter  more  in  detail  into  the 
various  theories  of  catalysis.  Still  it  seems  important  at  least  to  present 
in  a  few  words  one  of  these,  namely,  that  of  H.  v.  Euler.i  This  theory 
explains  the  mode  of  action  of  enzymes  and  the  inorganic  catalyzers  by 
assuming  an  increased  concentration  of  the  active  molecules  producing  the 
reaction,  i.e.,  by  increasing  the  ions  occurring  in  the  solution. 

The  action  of  enzymes  presupposes  the  presence  of  water,  and  the  best- 
studied  enzymotic   processes,   the   hydrolyses,   are  comparable  with  the 

+ 
action  of  acids  and  bases,  i.e.,  the  action  of  H  and  HO  ions.     In  the  hydroly- 
ses by  enzymes  an  activation  of  the  water  takes  place,  and  the  assumption 

+  - 

that  the  enzymes  act  by  an  increased  concentration  of  the  H  and  HO  ions, 

which  bring  about  the  reaction,  seems  to  be  attractive.  The  enzymes 
acting  analogously  to  mineral  acids  have  been  assumed,  according  to  this 

+ 
view,  to  be  producers  of  H  ions,  which  strongly  accelerate  cleavages  which 

would  otherwise  take  place  very  slowly  or  with  immeasurable  velocit}'. 
This  explanation  may,  as  developed  by  Friedenthal,^  be  applied  to  the 
oxidation  enzymes,  the  oxidases,  which  will  be  treated  of  later.  Water  is 
also  imperative  for  animal  oxidations,  and  the  reaction  of  the  fluid  is  in  this 
case  also  important  because  oxidations  are  regularly  accelerated  by  an 

alkaline  reaction,  i.e.,  by  the  presence  of  HO  ions.  We  can,  according  to 
Friedenthal,  consider  the  oxidases  as  producers  of  hydroxy  1  ions,  just  as 
we  can  consider  pepsin  as  a  producer  of  hydrogen  ions.  It  is  apparent  that 
this  view,  that  the  oxidases  are  producers  of  hydroxyl  ions,  is  in  harmony 
with  the  previously  mentioned  views  of  Traube  and  Nasse,^  that  the 
hydroxyl  ions  of  the  water  combine  with  the  oxidizable  substance. 

An  enzyme  is  an  organic  substance  formed  in  an  animal  or  plant  cell, 
which  is  destroyed  by  heating  its  aqueous  solution  and  which  acts  like  the 
catalyzers,  but  only  upon  certain  bodies.  Some  restriction  must  be  put 
to  this,  as  the  cells  do  not  always  produce  a  complete  enzyme,  but  oftener 
only    the    mother-substance    thereof.     These    mother-substances    of    the 


*  Zeitschr.  f.  physikal.  Chem.,  36.      "^  Salkowski's  Festschrift,  1904.     ^  See  pp.  5  and  6. 


16  INTRODUCTION. 

enzymes  are  called  proenzyines  or  zymogens.  The  zymogens  are  under 
certain  conditions  converted  into  enzymes,  and  in  certain  cases  this  is 
brought  about  by  the  special  action  of  bodies  called  kinases,  which  have 
been  little  studied  (see  Chapters  VI  and  IX), 

The  enzymes  are,  as  above  mentioned,  not  characterized  by  chemical 
reactions  in  the  ordinary  sense,  but  by  their  action.  From  this  stand- 
point most  of  the  enzymes  which  have  been  studied  can  be  divided  into 
two  chief  groups,  namely,  those  enzymes  having  a  hydrolytic  action  and 
those  having  an  oxidizing  action. 

Among  the  hydrolytic  enzymes  we  must  mention  in  the  first  place  the 
jyroteolytic  or  those  which  dissolve  proteid,  whose  representatives,  pepsin  and 
trypsin,  occur  in  the  animal  kingdom;  the  lipolytic  or  fat-splitting;  and 
the  amylolytic  or  diastatic  enzymes,  which  act  upon  the  starches.  In  this 
group  we  must  include  the  invertases,  which  split  the  disaccharides  into 
simpler  forms  of  sugar.  In  close  relationship  to  these  enzymes  we  may 
mention  the  ghicoside-splitting  enzymes,  which  occur  especially  in  the 
higher  plants.  Among  the  hydrolytic  enzymes  of  the  animal  kingdom 
we  must  also  include  arginase,  which  splits  arginineinto  urea  and  ornithine; 
the  two  desaniinating  enzymes  adenase  and  guanase,  which  convert  the  two 
bodies  adenine  and  guanine,  with  the  splitting  off  of  ammonia,  into  hypo- 
xanthine  and  xanthine  respectively;  and  the  hippuric-acid-splitting/iis^02;i/m 
and  the  urea-splitting  urease.  The  proteid-coagulating  enzymes,  chymosin 
■or  casein-coagvilating,  and  thrombin  or  blood-coagulating  enzyme,  belong  to 
a  special  though  not  clearly  defined  group. 

The  best-known  and  most  carefully  studied  enzyme  actions,  the  hydroly- 
ses,  are  exothermal  processes,  and  therefore  the  sum  of  the  new  products 
produced  has  a  lower  heat  of  combustion  than  the  original  substance. 
Now,  as  syntheses  are  generally  endothermal  reactions,  i.e.,  are  processes 
requiring  a  taking  up  of  heat  where  external  energy  must  be  supplied  before 
they  take  place,  and  also  as  the  enzymes  are  not  a  source  of  energy,  it 
used  to  be  generally  considered  that  the  enzymes  could  not  bring  about 
any  syntheses.  This  view  is  nevertheless  untenable,  and  it  has  also  been 
shown  that  enzymotic  hydrolyses  may  be  reversible  processes  which  pro- 
duce syntheses.  Croft  Hill  has  shown  that  maltase,  which,  as  is  well 
known,  has  a  splitting  action  upon  maltose,  also  has  the  power  of  regener- 
ating from  glucose  two  isomeric  bioses,  one  a  new  body  called  revertose  and 
another  which  is  probably  maltose  (see  also  Emmerling  i).  E.  Flscher 
and  E.  F.  Armstrong  ^  were  able  to  obtain  a  dissaccharide,  isolactose, 
from  galactose  and  glucose  by  means  of  kephir  lactase.     Hanriot,'  Kastle 

»  Hill,  Ber.  d.  d.  chem  Gesellsch.,  34,  and  Transactions  Chem.  Society,  1903,  83; 
Emmerling,  Ber.  d.  d.  chem.  Gesellsch.,  34. 
'  Ber.  d.  d.  chem.  Gesellsch.,  35. 
» Compt.  rend.,  132. 


ENZYME  ACTIONS.  17 

and  LoEVENHART  1  have  shown  that  the  Upases  can  bring  about  syntheses, 
and  finally  Emmerling  ^  has  been  able  to  synthesize  amygdalin  from  raan- 
delic-aeid-nitrile  glucoside  and  glucose  by  means  of  the  yeast  maltase. 
According  to  Abelous  and  Ribaut^  the  pig  and  horse  kidneys  contain 
an  enzyme  which  produces  hippuric  acid  from  benz^d  alcohol  and  glycocolL 
These  investigators  are  of  the  opinion  that  the  benzyl  alcohol  is  first  oxi- 
dized to  benzoic  acid  and  then  that  the  synthesis  is  brought  about  by  the 
aid  of  the  energy  set  free  in  this  process.  There  is  more  and  more  tendency 
to  accept  the  view  that  the  intracellular  enzymes,  which  will  be  discussed 
later,  are  of  importance  for  the  S3'ntheses  in  the  animal  body. 

The  second  group  of  enzymes  include  the  so-called  oxidation  fer-ments, 
which,  as  above  remarked,  are  recognized  as  of  great  importance  in  bringing 
about  oxidations  in  the  animal  body.  These  enzymes  do  not  all  act  in  the 
same  way,  and  correspondingly  we  differentiate  between  direct  oxidases 
or  oxidases  proper,  and  indirect  oxidases  or  peroxidases.  Certain  inves- 
tigators include  among  the  oxidation  enzymes  still  a  third  group,  the 
catalases,  which  split  peroxides  into  hydrogen  and  oxygen. 

Those  enzymes  which  transfer  oxj^gen  to  other  bodies  and  oxidize 
them  are  called  oxidases  or  direct  oxidases.  Peroxidases  or  indirect  oxi- 
dases are,  on  the  contrary,  enzymes  having  an  oxidizing  action  onl}^  in  the 
presence  of  hydroperoxides  or  another  peroxide,  as  they  decompose  the 
peroxide  and  bring  about  oxidation  by  the  oxygen  set  free.  Correspondingly 
the  oxidases  turn  tincture  of  giiaiacum  blue  directly,  while  the  peroxidases 
only  have  this  action  in  the  presence  of  a  peroxide.  The  catalases  do  not 
give  any  reaction  with  giiaiacum  either  directly  or  indirecth*  in  the  presence 
of  peroxides. 

According  to  the  investigations  of  Bach  and  Chodat  ■*  the  conditions  are 
otherwise.  According  to  the  observations  they  have  made  upon  plants, 
there  exist  no  oxidases  and  what  has  been  described  under  this  name  is 
only  a  mixture  of  oxygenases  and  peroxidases.  The  oxygenases  are 
of  a  protein  nature,  contain  manganese  or  iron,  and  are  converted  into 
peroxides  after  takmg  up  oxygen.  These  peroxides  themselves  have 
only  a  slight  oxidizing  power  but  are  made  active  by  the  peroxidases. 
The  peroxidases,  which  do  not  have  the  slightest  oxidizing  power  in  the 
absence  of  peroxides,  are  not  proteins.  In  oxidation,  according  to  the 
hypothesis  of  Bach  and  Chodat,  the  molecular  oxygen  is  first  converted 
by  the  oxygenase  into  peroxide.  This  peroxide  is  activated  by  the  peroxi- 
dase and  then  has  strong  oxidizing  power.     The  oxidizing  power  of  the 


*  Amer.  Chem.  Journ.,  24. 

2Ber.  d.  d.  chem.  Gesellsch.,  U,  3810. 

^  Compt.  rend.  Soc.  biol.,  52;   Maly's  Jahresber.,  30. 

*  Biochem.  Centralbl.,  1,  pp.  417  and  457. 


18  INTRODUCTION. 

so-called  direct  oxidases  is  brought  about  by  a  combined  action  of  the 
oxygenases  and  peroxidases. 

The  chemical  nature  of  the  oxidation  enzymes  is  still  unknown,  and 
the  statements  on  this  subject  are  very  contradictory.  Certain  oxidases 
are  supposed  to  be  nucleoproteids  (Spitzer),  others  globulins  (Abelous 
and  BiARNEs),  and  still  others,  like  the  liver  aldehydase  (Jacoby)  and 
laccase  (Bertrand),  are  of  a  non-protein  nature.  The  materials  upon 
which  the  oxidation  enzymes  act  may  also  be  very  different  from  each 
other.  Thus  the  oxidases  studied  by  Rohmann  and  Spitzer  may  Vjy 
synthetical  oxidation  produce  indophenol  from  a-naphthol  and  p-phenyl- 
enediamine  in  the  presence  of  alkali.  The  salicylase  or  aldehydase  detected 
in  the  liver  and  many  other  organs  oxidizes  many  aldehydes  to  their  cor- 
responding acids,  but  does  not  give  the  indophenol  reaction.  "The  laccase 
isolated  by  Bertrand  from  the  juice  of  the  lac -tree  has  an  oxidizing  action 
upon  polyhydric  p-phenols,  such  as  hydroquinone,  but  not  upon  tyrosine. 
The  bodies  called  tyrosinases,  first  found  by  Bertrand  *  in  certain  fungi 
and  later  also  found  by  Biedermann,  v.  Fijrth,  and  Schneider  in  the 
animal  kingdom,  have,  on  the  contrary,  an  action  upon  tyrosine,  converting 
it  into  homogentisic  acid  (Gonnermann  2)  or  other  colored  compounds. 
Another  oxidase  occurring  in  the  liver  and  spleen,  and  called  xanthine 
oxidase  by  Burian,  has  the  property,  as  shown  by  Spitzer,  Wiener, 
Schittenhelm,  and  Burian,^  of  transforming  xanthine  and  hypoxanthine 
into  uric  acid  by  oxidation. 

The  oxidases  and  peroxidases  as  well  as  the  catalases  occur  very  widely 
distributed  in  the  animal  and  plant  kingdoms. 

Like  other  enzymes,  the  oxidation  enzymes  show  also  a  pronounced 
specificity;  thus  a  certain  oxidase,  for  instance  laccase,  oxidizes  only  certain 
substances  and  not  others.  This  behavior,  which  is  difficult  of  explana- 
tion according  to  the  common  hypotheses  as  to  the  action  of  oxidation 
enzymes,  indicates,  according  to  Medwedew,^  that  in  the  oxidation  the 
active  substances  do  not  act  upon  the  oxygen,  but  rather  upon  the  sul- 
stance  to  be  oxidized.  We  cannot  at  present  give  any  statement  as  to 
the  extent  of  action  of  the  oxidation  enzymes  in  the  oxidations  of  the  ani- 
mal body,  and  it  is  still  a  question  whether  in  all  cases  where  oxidation 
enzymes  have  been  claimed  to  have  been  found  we  were  actually  dealing 
with  enzymes. 


'  In  regard  to  the  work  of  the  various  authors  cited,  see  foot-note,  p.  8. 

'  Biedermann,  Pfliiger's  Archiv,  72;  v.  Fiirth  and  Schneider,  Hofmeister's  Beitrage, 
1;   Gonnermann,  Pfliiger's  Archiv,  82. 

^Spitzer,  Pfliiger's  Archiv,  76;  Wiener,  Arch.  f.  exp.  Path.  u.  Pharm.,  42;  Schit- 
tenJielm,  Zeitschr.  f.  physiol.  Chem.,  42  and  43;  Burian,  ibid.,  43. 

*Pfliiger'.s  Archiv,  81. 


OXIDATION  ENZYMES.  19 

In  investigations  with  hydroperoxides  and  vegetable  peroxidases  Bach 
and  Chodat  ^  found  that  peroxides  and  peroxidases  always  took  part  in 
the  reaction  in  constant  proportions,  and  that  the  peroxidases  were  quickly 
used  up,  which  certainly  does  not  indicate  that  these  bodies  have  an  enzy- 
motic  nature.  Aso  ^  has  also  shown  that  in  certain  cases  where  an  apparent 
oxidase  action  was  present  ver}-  probably  we  w^ere  dealing  only  with  nitrites 
which  were  present;  and  finally,  attention  must  be  called  to  the  fact  that 
manganese  or  iron,  sometimes  in  considerable  amounts,  has  been  found 
in  many  oxidases.  As  manganous  and  ferrous  salts  are  active  as  cataly- 
zers in  certain  other  oxidations,  so  also  in  certain  cases  important  roles 
as'  oxygen-carriers  have  been  ascribed  to  these  metals,  for  instance  in 
laccase,  which  contains  manganese  (Bertrand),  and  the  oxidases  contain- 
ing iron  (Spitzer's  nucleoproteid).  Manchot^  by  his  work  on  the  auto- 
oxidation  of  ferrous  sulphate  has  called  attention  to  the  apparently  great 
importance  of  iron  for  physiological  oxidations,  and  the  work  of  Trillat,* 
who  has  prepared  colloidal  solutions  of  protein-manganese  which  had 
great  similarity  to  oxidase  solutions,  is  also  of  special  interest. 

Our  knowledge  of  the  reducing  enzymes,^  the  so-called  reductases  or 
hydrogenases,  is  even  still  more  meagre.  Certain  investigators  claim  that 
the  so-called  philothions,  which  develop  hydrogen  sulphide  in  the  presence 
of  sulphur  and  water,  belong  to  this  group,  while  others,  on  the  contrary-, 
do  not  accept  this  view  and  consider  the  enzymotic  nature  of  the  philothions 
as  doubtful.^  There  is  no  doubt  that  reductions  occur  to  a  great  extent 
in  the  animal  body  and  often  hand  in  hand  \nth  oxidations;  nevertheless 
the  question  as  to  how  far  special  reduction  enzymes  take  part  in  these 
reductions  is  still  an  open  one.  According  to  Abelous  and  Aloy  '  we  have 
indeed  enzymes  that  have  an  oxidizing  as  well  as  a  reducing  action,  for 
they  obtain  the  oxygen  necessary  for  the  oxidation  of  one  body  Ijy  remo^•ing 
it  from  another  substance  through  reduction. 

The  property  of  decomposing  h3^drogen  peroxide  has  been  observed  with 
many  enzymes,  but  this  property  does  not  Iselong  to  them,^  depending 

'  Ber.  d.  d.  chem.  CJesellsch.,  37. 

'  Beihefte  zum  botan.  Centralbl.,  18. 

'  Zeitschr.  f.  anorg.  Chem.,  27. 

'  Compt.  rend.,  137,  138. 

^  Abelous  and  Gerard,  Compt.  rend.,  129;   Pozzi-Escot,  Bull.  Soc.  chim.  (3),  27. 

•  De  Rey-Pailhade,  Recherches  exper.  sur  le  Philothion,  etc.,  Paris  (G.  Masson), 
1891,  and  Nouvelles  recherches  sur  le  Philothion,  Paris  (G.  Masson),  1892;  Pozzi-Escot, 
1.  c,  and  Chem.  Centralbl.,  1904,  1,  S.  1645;  Chodat  and  Bach,  Ber.  d.  d.  chem. 
Gesellsch.,  36;  Abelous  and  Ribaut,  Compt.  rend.,  137,  and  Bull.  Soc.  chim.,  Paris  (3), 
31. 

'  Compt.  rend.,  136,  137,  and  138. 

^  See  Al.  Schmidt,  Zur  Blutlehre,  Leipzig,  1892;  Jacobson,  Zeitschr.  f.  physiol. 
Chem.,  16. 


20  INTRODUCTION. 

rather  upon  another  enzyme,  a  catalase,  vhich  often  adheres  to  other 
enzymes  as  an  impurity.  The  catalases  were  first  closely  studied  by  O. 
LoEW,i  and  he  has  investigated  two  different  catalases — the  a-  and  /3-catalase. 
The  first,  which  is  not  soluble  in  water,  is  a  nucleoproteid,  while  the  other, 
^-catalase,  is  soluble  in  water  and  is  a  proteose. 

The  catalases,  whose  action  consists  in  decomposing  hydrogen  peroxide 
into  oxygen  and  hydrogen,  occur  widely  diffused  in  the  animal  and  plant 
kingdoms.  According  to  L.  Liebermaxn^  the  fatty  tissues  among  the 
animal  structures  seem  to  be  richest  in  catalases,  an  observation  which  has 
been  substantiated  and  developed  by  Euler.^  The  liver,  kidneys,  and 
spleen  are  relatively  rich  in  catalases,  while  the  brain  and  muscles  are 
poor  therein;  still  the  proportions  vary  somewhat  for  different  varieties 
of  animals.'*  As  has  long  been  known,  the  blood  also  contains  a  catalase, 
which  has  been  called  hcemase  by  Sexter.^ 

The  physiological  importance  of  the  catalases  is  still  unknown.  Ac- 
cording to  LoEW  ^  they  have  the  function  of  destroying  the  hydrogen  per- 
oxide, which  occurs  perhaps  as  an  intermediary  product  in  oxidations  and 
which  has  a  destructive  action  as  protoplasmic  poison;  but  this  assumption 
is  disputed  by  Euler  "^  and  others.  Euler  calls  attention  to  the  parallelism 
which  exists  between  the  fat-splitting  and  the  peroxide-splitting  action  of 
plant  and  animal  extracts  and  claims  that  the  lipolytic  extracts  have  the 
property  of  decomposing  hydrogen  peroxide. 

The  glycolytic  or  svigar-destroying  enzyme,  which  occurs  in  the  blood 
and  tissues  and  which  takes  part  in  the  decomposition  of  the  sugars,  stands 
in  close  relationship  to  the  oxidases.  We  will  discuss  this  enzyme  in  a 
following  chapter  in  speaking  of  glycosuria  and  the  question  of  diabetes, 
and  it  is  here  sufficient  to  remark  that  certain  investigators,  like  Spitzer, 
consider  this  enzyme  as  an  oxidase,  while  others,  on  the  contrary,  consider 
the  decomposition  of  the  sugar  in  the  tissues  to  be  a  process  analogous  to 
alcoholic  fermentation. 

Alcoholic  fermentation  by  means  of  yeast  or  zymase  is  not  an  oxidation 
in  the  ordinary  sense,  where  the  sugar  takes  up  free  oxygen.  It  is  rather 
an  internal  oxidation  where  a  part  of  the  molecule  is  oxidized  at  the  cost 

'  U.  S.  Dept.  of  Agriculture,  Rep.  68,  Washington,  1901,  and  Ber.  d.  d.  chem. 
Gesellsch.,  35. 

2  Pfliiger's  Arch.,  104. 

^  Hofmeister's  Beitrage,  7,  which  also  gives  the  references  to  the  literature. 

*  See  Battelli  and  Stern,  Compt.  rend.,  138;  BatteUi  and  HalifT,  Compt.  rend.  Soc. 
biol.,  o". 

^  Senter,  Zeitschr.  f .  physikal.  Chem.,  44,  also  A.  Jolles  and  Oppenheim,  Virchow's 
Arch.,  ISO;  Ville  and  Moitesnier,  Bull.  Soc.  chim.  (3),  29;  A.  Rosenbaum,  Salkow- 
ski's  Festschrift,  1904. 

•See  foot-note,  3,  p.  7. 

'  Hofmeister's  Beitrage,  7. 


GLYCOLYSIS   AND  ALCOHOL   FERMENTATION.  21 

of  another  part,  and  finally  a  destruction  into  alcohol  and  carbon  dioxide 
takes  place.  According  to  the  recent  investigations  of  Buchxer  and 
Meisexheimer,  Stoklasa,  and  Maze,^  we  are  dealing  here  with  the  united 
action  of  two  enzymes,  one  the  lactolase  (Stoklasa)  or  ladacidase  (Buch- 
xer and  ]\Ieisexheimer),  which  converts  the  sugar  into  lactic  acid,  while 
the  other,  the  zymase  (Buchxer  and  ]\Ieisexheimer)  or  alcoholase  (Stok- 
lasa), splits  the  lactic  acid  into  alcohol  and  carbon  dioxide.  According  to 
several  investigators,  the  sugar  passes  into  lactic  acid,  with  methylglyoxal, 
CH3.CO.CHO,  as  an  intermediary  body. 

Stoklasa  and  his  collaborators  ^  believe  that  an  alcoholic  fermentation 
b}'  means  of  a  zymase  or  perhaps  a  mixture  of  the  two  above-mentioned 
enzymes,  lactolase  and  alcoholase,  also  takes  place  in  animal  tissues.  Ob- 
jections to  these  investigations  have  been  made  by  several  experimenters 
who  claim  essentialh'  that  in  these  cases  we  are  dealing  only  with  the  action 
of  micro-organisms.^  Hammarstex  considers  that  the  views  of  Stoklasa 
and  his  collaborators  have  not  been  disproved,  and  one  cannot  exclude  the 
possibility  that  an  alcoholic  fermentation  may  also  occur  in  the  animal 
tissues  in  anaerobic  respiration. 

The  enzymes,  in  certain  instances,  may  also  act  upon  one  another,  and 
as  an  examj^le  of  this  kind  of  action  we  may  mention  Buchxer's  zymase, 
which  can  be  destroyed  by  the  proteolytic  enzyme  of  the  yeast-cells.  Pepsin, 
which  has  a  destructive  action  upon  diastases  and  especially  upon  trj'psin, 
is  another  example.  Of  special  interest  is  the  action  of  the  anti-enzymes 
upon  the  enzymes,  wliich  consists  in  retarding  or  arresting  the  specific 
action  of  the  enzyme  by  a  corresponding  anti-body.  This  subject  will  be 
discussed  later. 

Unfortunately  considerable  confusion  exists  in  the  nomenclature  of  the 
enzymes.  In  most  cases  the  enzyme  is  named  after  the  substance  upon  which 
it  acts,  thus  amylase,  lipase,  arginase,  urease;  in  other  cases  according  to  its 
action,  thus  oxidase,  reductase;  while  in  certain  cases  the  products  produced 
are  the  basis  for  the  name,  thus  alcoholase,  lactacidase,  glucase.  In  order  to 
obtain  a  clear  and  concise  nomenclature  of  the  enz}Tnes  v.  Lippmann  "  has  suggested 
that  we  construct  the  name  of  the  enzyme  out  of  two  words,  one  of  which  rep- 
resents the  substance  acted  upon  by  the  enzyme,  while  the  second  is  the  im- 
portant or  chief  product  produced  by  the  enzyme.  Thus  maltoglucase  is  an 
enzyme  which  produces  d-glucose  from  maltose,  amylmaltase  one  that  forms 
maltose  from  starch  (amylum),  etc. 

*  Buchiaer  and  Meisenheimer,  Ber.  d.  d.  chem.  Gesellsch.,  3"  and  38;  Stoklaha, 
Ber.  d.  d.  botan.  Gesellsch.,  22,  pp.  358  and  460;  Maze,  Compt.  rend.,  13S. 

^  Hofmeister's  Beitrage,  3;  Centralbl.  f.  Physiol.,  16,  17,  18;  Ber.  d.  d.  chem. 
Gesellsch.,  38;  see  also  Czemy,  ibid.,  36,  with  Jelinek,  Simacek,  and  Vitek,  Pfliiger's 
Arch.,  101. 

'  See  the  work  of  O.  Cohnheim,  Zeitschr.  f.  physiol.  Chem.,  39,  42,  43;  BatteUi, 
Compt.  rend.,  137;    Portier,  Compt.  rend.  Soc.  biol.,  57. 

*  Ber.  d.  d.  chem.  GeseUsch.,  36. 


22  INTRODUCTION. 

Many  enzymes  are  secreted  by  the  cells  as  such  or  as  proenzymes.  They 
act  outside  of  the  cells  in  which  they  were  formed,  or  they  act  after  having 
been  transformed  into  the  enzyme,  and  hence  are  called  secretion  enzymes 
or  e:xtracellular  enzymes. 

Besides  these  extracellular  enzymes  we  also  have  another  group  which 
act  within  the  cells,  hence  are  intracellular  and  therefore  are  called  intra- 
cellular enzymes  or  endoenzymes.  Numerous  enzymes  besides  the  yeast 
zymase  belong  to  this  group,  and  seemingly  also  oxidases  and  enzymes 
having  hydrolytic  action.  The  best  studied  of  this  group  are  the  proteo- 
lytic enzymes,  which  were  first  observed  by  Salkowski  and  his  pupils,  and 
which  bring  about  the  self-digestion  or  autodigestion  of  organs  in  the 
absence  of  micro-organisms.  This  autodigestion  has  been  the  subject  of 
numerous  investigations,  principally  by  the  Hofmeister  school  and  esj^e- 
cially  by  Jacoby.^  The  latter  has  given  the  name  autolysis  to  the  process, 
and  he  has  shown  that  the  enzymes  taking  part  in  this  action  do  not  come 
from  the  digestive  tract  and  are  not  pepsin  or  trypsin  taken  up  by  the 
cells.  In  autolysis  we  are  not  only  dealing  with  a  proteolysis,  but  several 
other  processes  occur,  such  as  the  splitting  of  fats  and  carbohydrates,  oxi- 
dations and  reductions,  and  perhaps  also  syntheses. 

We  therefore  generally  designate  as  autolysis  all  the  enzyme  actions 
which  take  place  in  removed  organs  or  fluids  without  the  aid  of  micro- 
organisms, but  it  must  not  be  forgotten  that  autolytic  processes  may  also 
ocour  intra  vitam  under  certain  conditions.  The  combined  action  of 
various  enzymes  in  autolysis  also  explains  to  us  why,  as  especially  shown  by 
Levene  and  by  Jones,^  the  products  obtained  by  the  hydrolytic  cleavage 
of  an  organ  by  means  of  an  acid  are  somewhat  different  from  those  products 
produced  on  autolysis. 

It  is  at  present  impossible  to  state  what  part  autolytic  processes  take  in 
life  under  physiological  conditions,  and  we  can  have  only  conjectures  on 
this  subject.  In  the  autolysis  of  a  removed  organ  or  of  one  through  which 
the  blood  is  not  flowing,  the  conditions  in  many  ways  are  quite  different 
from  the  conditions  in  life.  The  products  which  appear  after  weeks  or 
months  of  autolysis,  sometimes  in  very  small  quantities,  do  not  give  any 
clue  to  the  nature  of  the  processes,  and  conclusions  must  be  drawn  very 
carefully  from  these  results. 

The  post-mortem  autolyses,  as  far  as  studied,  are  chiefly  proteolyses, 
but  we  must  not  forget  that  the  enzymes  taking  part  are  in  many  cases 
most  active  in  acid  reaction,  while  they  have  only  a  weak  action  or  are 

'  A  complete  summary  of  the  literature  of  intracellular  enzymes  and  autolysis  may 
be  found  in  Jacoby,  Uber  die  Bedeutung  der  intrazellularen  Fermente,  etc.,  Ergeb- 
nisse  der  Physiologic,  Jahrg.  I,  Abt.  1,  1902. 

2  Levene,  Amer.  Joum.  of  Physiol.,  11  and  12,  and  Zeitschr.  f.  physiol.  Chem.,  41; 
"W.  Jones,  ibid.    42. 


AUTOLYSIS.  2a 

inactive  in  neutral  or  alkaline  reaction.  The  observations  of  Lane-Claypon 
and  ScHRYVER,^  that  the  autolysis  of  the  liver  and  kidney  begins  only  after 
a  latent  period  of  from  two  to  four  hours  subsequent  to  the  removal  of  the 
organ,  are  also  of  interest.  It  is  possible  that  this  is  due  to  the  fact  that  the 
enzymes  are  first  formed  from  the  proenzymes  after  the  death  of  the  organ, 
or  perhaps  certain  conditions  tending  to  retard  the  enzymotic  action  are 
removed.  Recent  investigations  of  Wiener  ^  show  that  the  post-mortem 
formation  of  acid  is  the  important  factor  in  this.  It  is  difficult  to  judge  of 
the  importance  of  the  autolytically  active  proteolytic  enzymes  for  the 
physiological  life  of  the  cells,  but  there  does  not  seem  to  be  any  doubt  as  to 
the  importance  of  these  enzymes  in  pathological  conditions. 

The  changes  of  the  liver  and  blood  in  acute  phosphorus  intoxication 
and  in  acute  yellow  atrophy  of  the  liver,  where  we  find  in  the  urine  the 
enzymotic  decomposition  products  of  the  proteins,  are  examples  of  an 
intra  vitam  autolysis  which  is  considered  by  some  as  an  abnormal  rise  in 
the  physiological  autolysis.  Another  example  is  the  solution  of  pneumonic 
infiltrations  by  the  enzymes  of  the  migrated  and  inclosed  leucocytes  as 
studied  by  Fr.  ]\Iuller  ^  and  this  is  at  the  same  time  an  example  of  hete- 
rolysis,  i.e.,  of  a  solution  or  a  destruction  in  an  organ  by  enzymes  not 
belonging  therein  but  introduced  from  without.  An  autol3'sis,  although  not 
very  marked,  occurs  in  those  organs  or  parts  of  organs  which  have  not 
been  normally  nourished  because  of  a  disturbance  in  the  circulation,  and 
they  are  gradually  consumed  by  this  action.  The  part  injured  undergoes 
solution,  while  the  healthy  part  remains  unattacked.  By  this  solvent 
action  as  well  as  by  the  formation  of  bactericidal  bodies,  as  observed  by 
CoNRADi,*  and  of  antitoxins  (Blum  ^)  by  means  of  autolysis,  we  can  consider 
this  autolysis  as  a  remedy  and  perhaps  also  as  a  protective  agent  for  the 
animal  body. 

For  the  present  it  is  impossible  to  judge  of  the  importance  of  the 
enzymes  active  in  autolysis  for  physiological  conditions,  but  this  does 
not  exclude  the  possibility  that  in  normal  cell  life  the  enzymes  play  a 
very  important  role.  Numerous  observations  show  this  to  be  true,  and 
we  tend  more  and  more  toward  the  view  that  the  chemical  transforma- 
tions in  the  living  cells  are  brought  about  by  enzymes  and  that  these  latter 
are  to  be  considered  as  the  chemical  tools  of  the  cells  (Hofmeister  and 
others  ^). 

*  Joum.  of  Physiol.,  31. 
'Centralbl.  f.  Physiol.,  19,  p.  349. 

'  Verhandl.  d.  naturforsch.  Gesellsch.  zu  Basel,  1901.  See  also  O.  Simon,  Deutsch. 
Arch.  f.  klin.  Med.,  1901. 

*  Hofmeister's  Beitrage,  1. 
^Ibid.,  5,  p.  142. 

*  F.  Hofmeister,  Die  chemische  Organisation  der  Zelle,  Braunschweig,  1901. 


24  INTRODUCTION. 

As  above  stated,  the  chemical  processes  in  animals  and  plants  do  not 
stand  in  opposition  to  each  other;  they  offer  differences  indeed,  but  still 
they  are  of  the  same  kind  from  a  qualitative  standpoint.  Pfluger  says 
that  there  exists  a  blood-relationship  between  all  living  cells  of  the  animal 
and  vegetable  kingdoms,  and  that  they  originate  from  the  same  root. 
The  animal  body  is  a  complex  of  cells,  hence  study  of  the  chemical  pro- 
cesses must  not  only  be  made  upon  higher  plants  but  also  upon  unicellular 
organisms  in  order  that  we  get  a  proper  explanation  of  the  chemical 
processes  in  the  animal  organism.  Although  a  biochemical  study  of  the 
micro-organisms  is  very  important,  we  must  bear  in  mind  also  the  important 
role  played  by  such  organisms  in  animal  life,  chiefly  as  exciters  of  disease; 
hence  the  study  of  the  conditions  of  life  of  these  micro-organisms  and  the 
products  produced  by  them  must  be  of  infuiite  importance  in  their  chemical 
investigation. 

The  products  produced  by  micro-organisms  may  be  of  very  different 
kinds.  Among  the  substances  produced  in  the  decomposition  of  animal 
fluids  and  tissues  by  putrefactive  organisms  we  find  those  having  a 
basic  nature.  To  this  class  belong  the  cadaver  alkaloids  called  ptomaines, 
first  found  by  Selmi  in  human  cadavers  and  then  specially  studied  by 
Brieger  and  Gautier.^  Certain  of  these  are  poisonous,  designated  as  tox- 
ines,  while  the  others  are  non-poisonous.  They  all  belong  to  the  aliphatic 
compounds  and  generally  do  not  contain  oxygen.  As  an  example  of 
these  basic  substances  we  must  mention  the  two  diamines,  cadaverine  or 
pentamethylenecUamine,  C5H14N2,  and  jmtrescine  or  tetramethylenediamine, 
C4H12N2,  which  have  awakened  special  interest  because  they  occur  in  the 
contents  of  the  intestine  and  in  the  urine  in  certain  pathological  condi- 
tions, especially  in  cholera  and  cj^stinuria.^  Among  the  bodies  produced 
by  putrefaction,  the  bacterial  poison  sepsine,  C5H14N2O2,  recently  isolated 
by  E.  Faust,^  is  of  especially  great  interest  because  to  this  substance  we 
ascribe  the  characteristic  toxic  action  of  putrefactive  masses.  Sepsine  was 
prepared  by  Faust  as  a  crystalline  sulphate  which  on  repeated  evaporation 
of  its  solution  was  readily  converted  into  cadaverine  sulphate. 

Those  substances  of  basic  nature  which  are  incessantly  and  regularly 
produced  as  products  of  the  decomposition  of  the  protein  substances  in 
the  living  organism,  and  which  therefore  are  to  be  considered  as  products 
of  the  physiological  metabolism,  have  been  called  leucomaines  by  Gautii:r 

'  Selmi,  Sulle  ptomaine  od  alcaloidi  cadaverici  c  loro  importanza  in  tossicologia, 
Bologna,  1878;  Ber.  d.  deutsch.  chem.  Gesellsch.,  11,  Correspond,  by  H.  S  hiff; 
Brieger,  Ueber  Ptomaine,  Parts  1,  2,  and  3,  Berlin,  1885-1886;  A.  Gautier,  Traite 
de  chimie  appliqu6e  k  la  physiologic,  2,  1873,  and  Compt.  rend.,  94. 

^  See  Brieger,  Berlin,  klin.  Wochenschr.,  1887;  Baumann  and  Udransky,  Zeitschr. 
f.  physiol.  Chem.,  13  and  15;  Brieger  and  Stadthagen,  Berlin,  klin.  Woohcnschr.,  1889. 

^  Arch.  f.  exp.  Path.  u.  Pharm.,  51. 


PTOMAINES  AND   LEUCO:\IAL\ES.  25 

in  contradistinction  to  the  ptomaines  and  toxines  produced  by  micro- 
organisms. These  bodies,  to  which  belong  several  well-known  animal 
extractives,  were  isolated  by  Gautier  ^  from  animal  tissues  such  as  the 
muscles.  The  hitherto  known  leucomaines,  of  which  a  few  are  poisonous 
in  small  amounts,  belong  to  the  choline,  the  uric  acid,  and  the  creatinine 
groups. 

The  leucomaines  are  considered  as  being  of  certain  importance  in  caus- 
ing disease.  It  has  been  contended  that  when  these  bodies  accumulate  on 
account  of  an  incomplete  excretion  or  oxidation  in  the  system,  an  auto- 
intoxication may  be  produced  (Bouchard  and  others  2). 

Of  especially  great  interest  are  the  toxines  which  are  found  in  the  higher 
plants  and  animals,  like  the  jequirity-bean  and  castor-seed,  in  the  poison 
of  snakes  and  spiders,  in  blood-serum,  etc.,  and  particularly  those  produced 
by  pathogenic  micro-organisms  which  have  an  unmistakable  relationship 
to  the  enz3-mes.  A  closer  study  of  these  various  bodies,  Ij-sines,  agglutinines, 
toxines,  etc.,  as  well  as  of  the  antitoxines  and  the  theory  of  immunit}',  does 
not  lie  within  the  scope  of  this  work,  and  although  the  subject  is  of  the 
greatest  importance,  it  cannot  be  treated  here.  We  can  only  call  atten- 
tion to  one  similarity  between  many  toxines  and  enzymes,  and  this  is 
important  in  connection  with  what  we  have  already  stated  in  regard  to 
the  enzymes.  As  by  the  repeated  introduction  of  a  toxine  into  an  animal 
body  we  can  excite  a  formation  of  the  corresponding  antitoxine,  so,  as  first 
shown  by  ^Iorgexroth,^  it  is  also  possible,  by  the  m creasing  introduction 
of  an  enzyme  (rennin,  for  example),  to  produce  an  antienzyme  (an  antiren- 
nin)  in  the  body.  Similar  antienzymes  have  been  produced  in  several 
other  cases,  but  this  is  not  suiprising,  as  this  is  onl}'  a  special  case  of  the 
general  immunity  theor}',  according  to  which  the  animal  bod}'  has  the 
power  of  making  foreign  substances  non-destructive  by  reaction  products 
formed  by  the  body. 

'  Bull.  Soc.  chim.,  43,  and  A.  Gautier,  Sur  les  alcaloides  derives  de  la  destruction 
bacterienne  ou  physiologique  des  tissus  animaux,  Paris,  1886. 

^  Bouchard,  Logons  sur  les  auto-intoxications  dans  les  maladies,  Paris,  1887.  See 
also  the  various  text-books  of  clinical  laedicine. 

^  Centralbl.  f.  Bakteriol.  u.  Parisitenkunde,  26. 


CHAPTER  II. 
THE   PROTEIN   SUBSTANCES. 

The  chief  mass  of  the  organic  constituents  of  animal  tissues  consists  of 
amorphous,  nitrogenized,  xery  complex  bodies  of  high  molecular  weight. 
These  bodies,  which  are  either  proteids  in  a  special  sense  or  bodies  nearly 
related  thereto,  take  first  rank  among  the  organic  constituents  of  the  ani- 
mal body  on  account  of  their  great  abundance.  For  this  reason  they  are 
classed  together  in  a  special  group  which  has  received  the  name  protein 
group  (from  npaorevo,  I  am  the  first,  or  take  the  first  place).  The  bodies 
belonging  to  these  several  groups  are  called  protein  substances,  although  in 
a  few  cases  the  protein  bodies  in  a  special  sense  are  designated  by  the 
same  name. 

The  several  protein  substances  ^  contain  carbon,  hydrogen,  nitrogen,  and 
oxygen.  The  majority  contain  also  sulphur,  a  few  phosphorus,  and  a  few 
also  iron:  Copper,  chlorine,  iodine,  and  bromine  have  been  found  in  some 
few  cases.  On  heating  the  protein  substances  they  gradually  decompose, 
producing  a  strong  odor  of  burnt  horn  or  wool.  At  the  same  time  they 
produce  inflammable  gases,  water, carbon  dioxide, ammonia,  and  nitrogenized 
bases,  besides  many  other  substances,  and  leave  a  large  quantity  of  carbon. 
On  hydrolytic  cleavage  they  all  yield,  besides  nitrogenous  basic  substances, 
especially  large  amounts  of  a-monamino-acids  of  different  kinds. 

The  nitrogen  occurs  in  the  protein  bodies  in  various  forms,  and  this  is 
also  revealed  in  the  division  of  the  nitrogen  among  the  cleavage  products. 
On  boiling  with  dilute  mineral  acids  we  obtain  (1)  so-called  amide  nitrogen, 
which  is  readily  split  off  as  ammonia;  (2)  a  guanidine  residue  which  is  com- 
bined with  diaminovalerianic  acid  as  arginine  and  which  has  also  been  called 
the  urea-forming  group ;  (3)  basic  nitrogen  or  diamino-acid  nitrogen,  which 
is  precipitated  by  phosphotungstic  acid  as  basic  products  (to  which  also 
the  guanidine  residue  of  arginine  belongs) ;    (4)   monamino-acid  nitrogen ; 

•See  "Eiweisskorper,"  Ladenburg's  Handworterbuch  der  Chemie,  3,  534-589, 
which  gives  a  very  complete  summary  of  the  Hterature  of  protein  substances  up  to 
1885.  The  more  recent  Hterature  up  to  the  year  1903  may  be  found  in  O.  Cohnheim, 
Chemie  der  Eiweisskorper,  Braunschweig,  1904.  See  also  Mann,  Chemistry  of  the 
Proteids,  London,  1906. 

26 


NITROGEN  OF  THE  PROTEINS.  27 

and  (5)  the  nitrogen  in  variable  amounts  which  appears  as  humus-like 
melanoidins,  which  seem  to  be  of  only  secondary  formation  as  products  of 
elaboration. 

The  quantitative  division  of  the  total  nitrogen  between  the  above 
five  groups  is  different  in  the  various  protein  substances,  and  moreover  can- 
not be  given  ^\ith  certainty,  because  of  the  above-mentioned  melanoidin 
formation  and  the  errors  in  the  methods  used.^  The  follo\Wng  gives  at  least 
an  approximate  idea  of  this  di^ision.2  The  loosely  combined  so-called  amide 
nitrogen  seems  to  be  entireh'  absent  in  the  protamines.  In  the  gelatines  we 
find  1-2  per  cent,  and  5-10  per  cent  in  other  animal  protein  substances;  in 
the  plant  gluten-proteids.  13-20  per  cent  of  the  total  nitrogen  is  amide  nitro- 
gen. The  guanidine  nitrogen  may  amount  in  the  protamines  to  22-44  per 
cent  of  the  total  nitrogen,  in  the  histones  to  12-13  per  cent,  in  the  gelatines 
about  8  per  cent,  and  in  the  other  protein  bodies  about  2-5  per  cent.  As 
basic  nitrogen  precipitable  by  phosphotungstic  acid  (including  the  guanidine 
residue)  we  find  35-88  per  cent  in  the  protamines,  35-42.5  per  cent  in  the 
histones,  15-25  per  cent  in  the  other  animal  protein  substances,  5-14  per 
cent  in  zein  and  the  gluten  proteid,  and  up  to  37  per  cent  in  the  plant 
globulins.  The  chief  quantity  of  the  nitrogen,  55-76  per  cent,  occurs,  ^ith 
the  exception  of  the  protamines,  as  the  monamino-acid  groups.  The  result-s 
for  the  melanoidin  nitrogen  vary  so  considerably  that  they  will  not  be 
mentioned. 

From  the  above  results  it  follows  that  the  nitrogen  of  most  protein 
bodies  exists  in  such  combination  that  the  chief  quantity  appears  in  the 
cleavage  products  as  amino-compounds  on  hydrolytic  cleavage  by  acids. 
By  the  action  of  nitrous  acid  upon  proteins  only  a  xery  small  part,  1-2  per 
cent,  of  the  nitrogen  is  evolved,^  which  seems  to  indicate  that  NII2  groups 
exist  only  to  a  slight  extent  in  protein  substances.  This  assumption  does 
not  have  sufficient  foundation,  for,  according  to  Levites,^  the  quantity  of 
amide  nitrogen  is  not  diminished  by  the  action  of  nitrous  acid  upon  the 
protein  substances.  In  ^^ew  of  several  observations,  it  is  generally  ad- 
mitted that  the  amino-groups  occurring  in  the  cleavage  products  exist  in 
the  original  protein  substance  chiefly  as  imino-groups. 

The  sulphur  occurs  in  the  different  protein  bodies  in  very'  different 


*  See  the  work  of  Hausmann,  Zeitschr.  f.  physiol.  Chem.,  27  and  29;  Henderson, 
ibid.,  2";  Kossel  and  Kutscher,  ibid.,  30;  Kutscher,  ibid.,  31,  38;  Hart,  ibid.,  33; 
Giimbel,  Hofmeister's  Beitrage,  0;   Rothera,  ibid. 

^  See  the  works  given  in  foot-note  1  and  Blum,  Zeitschr.  f.  physiol.  Chem.,  30; 
Kossel,  Ber.  d.  d.  chem.  Gesellsch.,  34,  3214;  Hofmeister,  Ergebnisse  der  Physiol., 
Jahrg.  I,  Abt.  1,  759,  which  also  contains  the  literature;  Osborne  and  Harris,  Joum. 
Amer.  Chem.  Soc,  25;   and  Giimbel,  1.  c. 

2  See  C.  Paal,  Ber.  d.  d.  chem.  GeseUsch.,  29;  H.  SchifT,  ibid.,  1354;  O.  Loew, 
Chemiker  Zeitung,  1896;   and  O.  Xasse,  Pfliiger's  Arch.,  6. 

^  Levites,  Zeitschr.  f.  physiol.  Chem.,  43. 


28  THE  PROTEIN  SUBSTANCES. 

amounts.  Certain  of  them,  such  as  the  protamines  and  apparently  also 
certain  bacterial  proteids/  are  free  from  sulphur;  some,  such  as  gelatine 
and  elastin,  are  very  poor  in  sulphur;  while  others,  especially  horn  sub- 
stances, are  relatively  rich  in  sulphur.  On  hydrolytic  cleavage  with  min- 
eral acids,  the  sulphur  of  the  protein  substances  is  regularly,  at  least  in 
part,  split  off  as  cystine  (K.  ^Iorner)  or,  with  bodies  poorer  in  sulphur,  as 
cystein  (Embden),  but  this,  according  to  ^Morn'er  and  Patten,  is  a  second- 
ary^ formation.  From  certain  protein  substances  a-thiolactic  acid  (Suter, 
Friedmann,  Frankel),  wliich]\IORXER  claims  is  also  produced  secondarily, 
mercaptans  and  sulphuretted  hydrogen  (Sieber  and  Schoubenko,  Rub- 
xer),  and  a  body  having  the  odor  of  ethyl  sulphide  (Drechsel)  have  been 
obtained  .2 

A  part  of  the  sulphur  separates  as  potassium  or  sodium  sulphide  on 
boiling  with  caustic  potash  or  soda,  and  may  be  detected  by  lead  acetate 
and  quantitatively  determined  (Fleitmann,  Daxilewsky,  Kruger,  Fr. 
ScHULZ,  Osborne,  K.  ^Morner^).  What  remains  can  be  detected  only 
after  fusing  with  potassium  nitrate  and  sodium  carbonate  and  testing  for 
sulphates.  The  ratio  between  the  sulphur  spUt  off  b}'  alkali  and  that  not  sj^lit 
off  is  different  in  various  proteins.  No  conclusions  can  be  dra\\ai  from 
this  in  regard  to  the  number  of  forms  of  combination  which  the  sulphur 
has  in  the  protein  molecule.  As  shown  by  K.  ]\Iorxer,  onh'  about  three- 
fourths  of  the  sulphur  in  cystine  can  be  split  off  by  alkali,  and  the  same 
is  true  for  the  cystine-yielding  complex  of  the  protein  substances.  If  the 
quantity  of  lead-blackening  sulphur  in  a  protein  body  be  multiplied  by 
I,  we  obtain  the  quantity  corresponding  to  the  cystine  sulphur  in  the  body. 
By  such  calculation  ]Morxer  found  in  certain  bodies,  such  as  horn  sub- 
stance, seral])umin  and  serglobulin,  that  the  quantity  of  cystine  sulphur  and 
total  sulphur  were  identical,  and  therefore  we  have  no  reason  for  consider- 
ing the  sulphur  in  these  bodies  as  existing  in  more  than  one  form  of  com- 
bination. In  other  proteins,  such  as  fibrinogen  and  ovalbumin,  on  the 
contrary,  only  one-half  or  one-third  of  the  sulphur  appeared  as  cystine 
sulphur. 

According  to  Raikow  ■*  keratin-like  proteins  split  off  sulphur  dioxide  on 

'  See  Nencki  and  Schaffer,  Joum.  f.  prakt.  Chem.  (N.  F.),  20,  and  M.  Nencki, 
Ber.  d.  d.  chem.  GeselLsch.,  17. 

2  K.  Morner,  Zeitschr.  f.  physiol.  Chem.,  28,  34,  and  42;  Patten,  ibid.,  89;  Embden, 
ibid.,  32;  Suter,  ibid.,  20;  Friedmann,  Hofmeister's  Beitrage,  3;  Sieber  and  Schou- 
benko, Archiv  d.  sciences  biol.  de  St.  Petersbourg,  1;  Rubner,  Arch.  f.  Hygiene,  19; 
Drechsel,  Centralbl.  f.  Physiol.,  10,  529;  Frankel,  Sitzungsber.  d.  Wien.  Akad.,  112, 
II  b,  K03 

'  Fleitmann,  Annal.  der  Chem.  und  Pharm.,  60;  Danilcwsky,  Zeitschr.  f.  physiol. 
Chem.,  7;  Kriiger,  Pfliiger's  Archiv,  43;  F.  Schulz,  Zeitschr.  f.  physiol.  Chem.,  25; 
Osborne,  Connecticut  Agric.  Expt.  Station  Report  1000;  Morner,  1.  c. 

^  See  Biochem.  Centralbl.,  4,  p.  353. 


CLEAVAGE   PRODUCTS   OF   THE   PROTEINS.  29 

treatment  witli  phosphoric  acid  at  ordinan*  temperatures;  hence  it  follows 
that  a  part  of  the  sulj^hiir  in  the  proteins,  esi:)ecially  in  the  keratins,  exists 
in  direct  combination  ^\'ith  oxygen  and  probably  combined  as  in  the 
sulphites. 

The  constitution  of  the  protein  bodies  is  still  unknown,  although  the 
great  advances  made  in  the  last  few  yesLTs  have  brought  us  essentially 
closer  to  the  elucidation  of  the  question.  In  studying  the  constitution  of 
the  protein  bodies  they  have  been  broken  up  in  various  ways  into  simpler 
portions,  and  the  methods  used  for  this  purpose  have  been  of  different 
kinds.  In  such  decompositions,  for  which  the  proteids  in  the  true  sense  have 
been  primarily  used,  esjiecially  those  that  can  be  prepared  in  the  cr^'stalline 
form,  first  large  atomic  complexes — proteo-ses  and  peptones — are  obtained 
which  still  have  protein  characteristics,  and  these  then  suffer  further  de- 
composition until  finally  we  obtain  simpler,  generally  cr\-stalline,  or  at 
least  well  characterized  end  products. 

On  heating  protein  T\-ith  barium  hydrate  and  water  m  sealed  tubes  to 
Io0-2o0°  C.  ScHUTZEXBERGER  ^  obtauied  a  mixture  of  products  among 
which  were  ammonia,  carbon  dioxide,  oxalic  acid,  acetic  acid,  and,  as  chief 
product,  a  mixture  of  amino-acids.  The  conclusion  he  drew  from  this 
experiment,  that  the  proteid  is  a  complex  ureide  or  oxamide,  cannot  be  con- 
sidered for  several  reasons  .2 

On  fusing  proteins  with  caustic  alkali  we  obtain  ammonia,  methyl  mer- 
captan,  and  other  volatile  products;  also  leucine,  from  which  then  volatile 
fatty  acids,  such  as  acetic  acid,  valerianic  acid,  and  also  butyric  acid  are 
obtamed,  and  also  tvrosine,  from  which  latter  phenol,  indol,  and  skatol  are 
produced.  As  to  the  products  prepared  by  hydrolytic  cleavage  ^dth  mm- 
eral  acids  we  have  a  number  of  investigations  by  various  experimenters, 
especialh^  Hlasiwetz  and  Habermanx,  Ritthausex  and  Kreusler.  E. 
ScHULZE  and  his  collaborators,  Drechsel,  Siegfried,  R.  Cohx,  Kossel 
and  his  pupils,  K.  Morxer,  Abderhaldex,  Skraup,  and  recently  E.  Fis- 
cher and  his  collaborators.^  The  chief  products  thus  obtained  are  mon- 
amino-acids,  such  as  glycocoll,  alanine,  amino  valerianic  acid,  leucine,  tyro- 
sine, phenylaminopropionic  acid,  aspartic  and  glutamic  acids,  cysteine  and  its 
sulphide  cystine;  the  so-called  hexone  bases,  lysine,  arginine,  and  histidine, 
of  which  the  first  two  are  diamino-acids;  oxymonamino-acids,  such  as  serine, 
oxyaminosuccinic  acid,  and  oxyaminosuberic  acid;  ox^-diamino-acids,  such 
as  oxydiaminosuberic  acid,  ox^^diaminosebacic  acid,  diaminotrioxvdodeca- 


'  -Ajinal.  de  chim.  ct  phys.  (5),  16,  and  Bull.  Soc.  chim.,  23  and  24. 

^  See  Habermann  and  Ehrenfeld,  Zeitschr.  f.  physiol.  Chem.,  30. 

^  In  regard  to  the  literature  see  O.  Cohnheim,  Chemie  der  Eiweisskorper,  Braun- 
schweig, 1904,  and  F.  Hofmcister,  Ergebnisse  der  Phy.siologie,  Jahr^.  I,  Abt.  1,  7.59, 
1902;  E.Fischer,  Untersuchungen  iiber  Animosauren,  Polypeptide  und  Proteine  (1899-- 
1906),  Berlin,  1906;  also  Mann,  Chemistry  of  the  Proteids,  London,  1906.  See  also 
special  references. 


30  THE  PROTEIN  SUBSTANCES. 

noic  acid,  caseanic  and  caseinic  acids;  a-pyrrolidine  and  oxypyrrolidine  car- 
boxylic  acids;  indolaminopropionic  acid;  sulphuretted  hydrogen,  ethyl 
sulphide,  leucinimide,  ammonia,  and  melanoidins,^  which  latter  seem  to  be 
secondary  condensation  products. 

The  proteins  can  be  split  into  a  large  number  of  bodies  by  the  proteo- 
lytic enzymes,  and  these  will  be  presented  later.  In  the  first  place  proteoses 
and  peptones  are  produced,  also  an  abundance  of  monamino-acids  of  dif- 
ferent kmds,  hexone  bases,  tr^^ptophane  (proteinochromogen),  which  is 
indolaminopropionic  acid,  and  finally  oxyphenylethylamine,  diamines,  and 
a  little  ammonia  and  other  substances. 

A  great  many  substances  are  produced  in  the  putrefaction  of  proteins. 
First  the  same  bodies  as  are  formed  in  the  decomposition  by  means  of 
proteolytic  enzymes  are  produced,  and  then  a  further  decomposition  occurs 
with  the  formation  of  a  large  number  of  bodies  belonging  in  part  to  the 
aliphatic  and  in  part  to  the  aromatic  and  heterocyclic  series.  Of  the  first 
series  we  have  ammonium  salts  of  volatile  fatty  acids,  such  as  caproic, 
valerianic,  and  butyric  acids,  also  succinic  acid,  carbon  dioxide,  methane, 
hydrogen,  sulphuretted  hydrogen,  methyl  mercaptan,  and  others.  The 
ptomaines  also  belong  to  these  products,  and  are  probably  in  part  formed 
by  very  different  chemical  processes,  or  even  syntheses. 

E.  Salkowski  divides  the  putrefactive  jDroducts  of  the  aromatic  and 
heterocyclic  series  into  three  groups :  (a)  the  phenol  group,  to  which  tyrosine, 
the  aromatic  oxy acids,  phenol,  and  cresol  belong;  (b)  the  phenyl  group, 
including  phenylacetic  acid  and  phenylpropionic  acid;  and  lastly  (c)  the 
indol  group,  which  includes  indol,  skatol,  skatolacetic  acid,  and  skatolcar- 
boxylic  acid.  These  various  products  are  formed  during  putrefaction  with 
access  of  air.  Nencki  and  Bovet^  obtained  only  p-oxyphenylpropionic 
acid,  phenylpropionic  acid,  and  skatolacetic  acid  on  the  putrefaction  of 
proteins  by  anaerobic  schizomycetes  in  the  absence  of  oxygen.  These  three 
acids  are  produced  by  the  action  of  nascent  hydrogen  on  the  corresponding 
amino-acids,  namely,  tyrosine,  phenylaminopropionic  acid,  and  skatolamino- 
acetic  acid  (indolaminopropionic  acid),  and  according  to  Nencki  these  three 
last-mentioned  amino-acids  exist  preformed  in  the  protein  molecule. 

By  the  moderate  action  of  chlorine,  bromine,  or  iodine  upon  proteins  these 
halogens  enter  into  more  or  less  firm  combination  with  the  molecule  (Loevv, 
Blum,  Blum  and  Vaubel,  Liebrecht,  Hopkins  and  Brook,  Hofmeister, 
KuRAjEFF,  and  others),  and  according  to  the  method  of  procedure  we  can 
prepare  derivatives  having  different  but  constant  amounts  of  halogens  (Hop- 
kins and  PixKUs).  The  proteins  are  so  changed  that  they  do  not  split  off 
sulphur  on  treatment  with  alkali,  nor  do  they  respond  to  Millon's  reaction, 


'  See  Samuely,  Hofmeister's  Beitrage,  2. 

'  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  12,  215,  and  27,  302;    Nencki  and   Bovet, 
Monatshefte  f,  Chem.,  10. 


OXIDATION   PRODUCTS  OF  THE  PROTEINS.  31 

nor  do  they  yield  tyrosine  as  a  cleavage  product.  This  is  ordinarily  ex- 
plained by  the  supposition  that  a  substitution  of  hydrogen  by  iodine  takes 
place  in  the  aromatic  t3-rosine  nucleus;  but  since  according  to  Oswald  the 
heteroproteoses,  which  yield  only  vers-  little  tyrosine,  take  up  about  the 
same  quantity  of  iodine  as  the  protoproteoses,  which  yield  considerable 
tyrosine,  it  appears  that  the  iodine  is  united  to  other  groups  besides  the 
tyrosine-yielding  atomic  complex.  By  the  action  of  iodine  an  oxidation  alsc^ 
occurs,  and  Schmidt  ^  has  shown  that  a  continuous  splitting  off  of  amino- 
groups  takes  place.  According  to  him  phenol  and  p-cresol,  cleavage  prod 
ucts  of  tj^rosine,  besides  benzoic  acid,  are  produced  by  the  oxidation  of 
phenylaminopropionic  acid. 

By  the  oxidation  of  proteid  by  means  of  potassium  permanganate 
Maly  obtained  an  acid,  oxyprotosulphonic  acid,  C  51.21,  H  6.89,  N  14.59,. 
S  1.77,  O  25.54  per  cent,  which  is  not  a  cleavage  product,  but  an  oxida- 
tion product  in  which  the  group  SH  is  changed  into  SOo.OH.  This  acid 
does  not  give  the  proper  color  reaction  with  Millon's  reagent,  yields  no 
tyrosine  or  indol,  but  gives  benzene  on  fusing  with  alkali.  On  continued 
oxidation  Maly  obtained  another  acid,  peroxyproteic  acid,  which  gives  the 
biuret  reaction,  but  is  not  precipitated  by  most  protein  precipitants.  The 
oxyprotein  obtained  by  Schulz  on  the  oxidation  of  proteid  by  hydrogen 
peroxide  is  closely  related  to  oxyprotosulphonic  acid  in  composition  and 
general  characteristics,  but  contains  lead-blackening  sulphur  and  gives 
Millon's  reaction.  The  oxyprotein  is  claimed  to  be  a  pure  oxidation 
product,  while  in  the  production  of  oxyprotosulphonic  acid  Schulz  claims 
that  a  cleavage  takes  place.  According  to  the  recent  investigations  of 
V.  FuRTH^  there  exist  at  least  three  different  peroxyproteic  acids  (from 
casein)  which  differ  from  each  other  by  a  different  division  of  the  nitrogen 
in  the  molecule.  On  treatment  with  baryta-water  we  find  that  they  split 
off  basic  complexes  and  oxalic-acid  groups,  and  new  bodies,  the  desamino- 
proteic  acids,  which  give  the  biuret  reaction,  are  produced.  These  acids, 
which  on  hydrolysis  give  benzoic  acid  but  no  diamino-acids,  ma}^  be  further 
oxidized,  which  is  not  true  of  the  peroxyproteic  acids,  and  yield  a  new 
group  of  acids,  the  kyroproteic  acids,  which  give  the  biuret  reaction,  hold 
about  one-half  of  their  nitrogen  (11.08  per  cent  total  nitrogen)  in  acid-amide- 
like combination,  but  yield  neither  basic  products  nor  benzoic  acid. 


•  Loew,  Joum.  f.  prakt.  Chem.  (N.  F.),  31;  Blum,  Miinch.  med.  Wochenschr., 
1896j  Blum  and  Vaubel,  Joum.  f.  prakt.  Chem.  (N.  F.),  5";  Liebrecht,  Ber.  d.  deutsch. 
chem.  Gesellsch.,  30;  Hopkins  and  Brook,  Joum.  of  Physiol.,  22;  Hopkins  and  Pin- 
kus,  Ber.  d.  deutsch.  chem.  Gesellsch.,  31;  Hofmeister,  Zeitschr.  f.  physiol.  Chem., 
24;  Kurajeff,  ibid.,  26;  Oswald,  Hofmeister's  Beitrage,  3;  C.  H.  L.  Schmidt,  Zeitschr. 
f.  physiol.  Chem.,  35,  38,  37. 

2  Maly,  Sitzungsber.  d.  k.  Akad.  d.  Wissensch.  Wien,  91  and  97.  Also  Monatshefte  f. 
Chem.,  6  and  9.  See  also  Bondzjaiski  and  Zoja,  Zeitschr.  f.  physiol.  Chem.,  19; 
Bemert,  ibid.,  26;  Schulz,  ibid.,  29;  v.  Furth,  Hofmeister's  Beitrage,  6. 


32  THE  TROTEIN  SUBSTANCES. 

On  the  oxidation  of  gelatine  or  proteid  witli  permanganate  we  obtain 
also  oxaminic  acid,  oxamide,  oxalic  acid,  oxaluric-acid  amide,  succinic  acid, 
several  volatile  fatty  acids,  and  giianidine,  which  was  first  shown  liy  Lossen 
as  an  oxidation  product  (Kutscher,  Zickgraf,  Seemaxx.  Kutscher  and 
Schexck).^ 

On  the  oxidation  of  gelatine  by  ferrous  sulphate  and  hydrogen  peroxide 
Blumenthal  and  Neuberg  have  obtained  acetone  as  a  product,  and  Orgler  -  the 
same  from  ovalbumin.  Jolles  '  claims  to  have  obtained  large  ciuantities  of  urea 
in  the  oxidation  of  various  proteins  by  potassium  permanganate  in  acid  solu- 
tion, but  this  has  been  disputed  by  other  investigators.  On  the  oxidation  of 
protein  in  acid  liquids,  volatile  fatty  acids,  their  aldehydes,  nitriles  and  ketones, 
also  hydrocyanic  acid,  benzoic  acid,  and  other  bodies,  have  been  obtained. 

Nitric  acid  gives  various  nitro-products.  A  melanoidin  substance,  xaniho- 
}nela>u)i,  has  been  obtained  by  v.  FVrth.*  Habermanx  and  Ehhexfeld  ^ 
also  obtained  oxyglutaric  acid  among  other  products.  By  the  action  of  bromine 
under  strong  pressure  a  number  of  products  have  been  obtained:  bromanil  and 
tribromacetic  acid,  bromoform,  leucinimide,  leucine,  oxalic  acid,  tribromamino- 
benzoic  acid,  and  other  bodies.  With  aqua  regia,  fumaric  acid,  oxalic  acid,  chor- 
iizol,  and  other  bodies  are  obtained.  The  recent  investigations  of  Habermanx 
^nd  Ehrexfeld  and  Panzer  '^  upon  the  action  of  chlorine  upon  jiroteins  and 
closely  related  products  are  important. 

By  the  dry  distillation  of  proteins  we  obtain  a  large  number  of  decomposition 
products  having  a  disagreeable  burnt  odor,  and  a  porous  glistening  mass  of  carbon 
containing  nitrogen  is  left  as  a  residue.  The  products  of  distillation  are  partly 
an  alkaline  liquid  which  contains  ammonium  carbonate  and  acetate,  ammonium 
sulphide,  ammonium  cyanide,  an  inflammable  oil,  and  other  bodies,  and  a  brown 
oil  which  contains  hydrocarbons,  nitrogenized  bases  belonging  to  the  aniline  and 
pyridine  series,  and  a  number  of  unknown  substances. 

The  occurrence  of  protein  substances  which  contain  a  carbohydrate 
group  has  been  known  for  a  long  time.  The  nature  of  this  carbohydrate, 
which  can  be  split  off  by  acid  and  which  may  amount  to  as  much  as  35 
per  cent,  has  been  explained  chiefly  by  the  investigations  of  Friedrich 
^MfLLER  "^  and  his  students.  They  have  shown  that  it  is  always  an  amino- 
sugar  and  generalh'  glucosamine.  That  so-called  true  proteids  also  }'ield 
a  carbohydrate  on  hydrolytic  cleavage  was  first  shown  by  Pavy,  using 
ovalbumin.     The  continued  investigations  of  Fr.  Muller,  Weydemaxx, 


'Lossen,  Annal.  d.  Chem.  u.  Pharm.,  201;  Kutscher,  Zeitschr.  f.  physiol.  Chem., 
32;  Zickgraf,  ibid.,  41;  Seemann,  ibid.,  44;  Kutscher  and  Schenck,  Ber.  d.  d.  chem. 
GeselLsch.,  3"  and  38. 

^  Bhimenthal  and  Neuberg,  Deutsch.  med.  Wochenschr.,  1901;  Orgler,  Hofmeister's 
Beit  rage,  1. 

^  Zeitschr.  f.  physiol.  Chem.,  32  and  38. 

''Sec  Maly's  Jahresbcr.,  30,  24. 

^  Zeitschr.  f.  physiol.  Chem.,  35. 

*  Hahermann  and  Ehrenfeld,  ibid.,  Panzer,  ibid.,  33  and  34. 

'  iiiiller,  Sitzungsber.  d.  Ges.  d.  Naturw.  zu  Marburg,  1896  and  1898,  and  Zeitschc 
f.  Biologie,  42. 


CARBOHYDRATE  MOIETY  OF  THE  PROTEINS.       33 

Seemann,  Frankel,  Hofmeister,  and  Laxgsteix  ^  have  demonstrated 
that  in  these  cases  the  carbohydrate  is  also  glucosamine.  A  carbohydrate 
complex,  although  sometimes  only  to  a  very  slight  amount,  has  also  been 
det€cted  in  other  proteins,  ovoglobulin,  serglobulin,  seralbumin,  pea- 
globulin,  albumin  of  the  graminese,  yolk-proteid,  and  fibrin.  In  other 
proteins,  on  the  contrary,  such  as  edestin  (of  the  hemp-seed)  and  casein, 
myosin,  pure  fibrinogen,  and  ovovitellin,  carbohydrates  have  been  sought 
for  with  negative  results.  All  proteins  hence  do  not  contain  a  carbohy- 
drate group,  and  future  investigators  must  therefore  decide  whether  the 
carbohydrate  groups  belong  positively  to  the  protein  complex  or  whether 
they  are  united  with  the  protein  only  as  impurities.  Several  observa- 
tions ^  show  that  in  working  with  crystalline  proteins  a  contamination  with 
other  protein  substances  is  unfortunately  not  excluded,  and  this  must  not 
be  lost  sight  of,  especially  as  the  quantity  of  carbohydrates  obtained  is 
often  very  small.  In  the  present  state  of  our  knowledge  we  are  not  war- 
ranted in  considering  the  carbohydrate  groups  as  belonging  to  the  carbon 
nucleus  produced  on  the  destruction  of  the  real  protein  complex. 

The  previously  mentioned  methods  used  in  studying  the  structure 
of  the  protein  substances  are  not  of  the  same  value,  but  they  in  part  sub- 
stantiate each  other.  Of  these  we  must  mention  the  hydrolysis  by  means 
of  boiling  dilute  mineral  acids,  or  by  proteolytic  enzymes,  as  the  best 
methods  for  obtaining  the  carbon  nuclei  in  the  protein  molecule.  The 
most  important  of  the  carbon  nuclei  obtained  are  as  follows: 

I.    The  Nuclei  belonging  to  the  Aliphatic  Series. 

A.  Sulphur  free,  but  containing  nitrogen:  1.  A  guanidine  residue  (combined  with. 
ornithine  as  arginine).  2.  Monobasic  monamiiio-acids:  Glycocoll  (aminoacetic 
acid),  alanine  (aminopropionic  acid),  aminovalerianic  acid,  leucine  (isobutylamino- 
acetic  acid),  and  isoleucine.  3.  Bibasic  monamino-acids:  Aspartic  acid  (amino- 
succinic  acid)  and  glutamic  acid  (aminoglutaric  acid).  4.  Oxymonamino-acids: 
serine  (oxyaminopropionic  acid)  oxyaminosuccinic  acid  and  oxyaminosuberic 
acid.  5.  Monobasic  diamino-acids:  Diaminoacetic  acid,  ornithine  (diaminovaleri- 
anic  acid)  and  lysine  (diaminocaproic  acid).  6.  Oxy diamino-acids:  Oxydiamino- 
suberic  acid,  oxydiaminosebacic  acid,  diaminotrioxydodecanoic  acid,  caseanic  and 
caseinic  acids. 

B.  Sulphurized:  Cysteine  (aminothiolactic  acid)  and  its  sulphide  cystine,  thio- 
lactic  acid,  mercaptans,  and  ethyl  sulphide. 

II.    The  Nuclei  belonging  to  the  Carbocyclic  Series. 
Phenylaminopropionic  acid  and  tyrosine. 

'  In  regard  to  the  literature  on  this  subject  see  the  work  of  Fr.  Miiller,  Zeitschr. 
f.  Biologie,42,  and  Langstein,  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1,63,  Zeitschr. 
f.  physiol.  Chem.,  41,  and  Hofmeister's  Beitrage,  6.  See  also  Abderhalden,  Bergell, 
and  Dorpinghaus,  Zeitschr.  f.  physiol.  Chem..  41. 

'See  Wichmann,  Zeitschr.  f.  physiol.  Chem.,  23,  and  N.  Schulz,  Die  Grosse  des 
Eiweissmolekiils,  Jena,  1903,  51. 


34  THE  PROTEIN  SUBSTANCES. 


III.    The  Nuclei  belonging  to  the  Heterocyclic  Series. 

A.  Oi  the  pyrrol  group:  Pyrrolidine  carboxylic  acid  (n-proline)  and  oxypyrro- 
lidine  carboxylic  acid. 

B.  Of  the  indol  group:  Tryptophane  or  indolaminoproijionic  acid,  from  which 
iiidol  and  skatol  are  produced  by  putrefaction. 

In  regard  to  these  carbon  nuclei  it  must  be  remarked  that  they  are 
not  all  found  in  every  protein  body  thus  far  investigated,  and  also  that 
one  and  the  same  cleavage  product,  such,  for  example,  as  glycocoll,  leu- 
cine, tyrosine,  or  cystine,  is  obtained  in  very  variable  amounts  from  differ- 
ent protein  substances.  It  is  very  difhcult  to  say  to  what  extent  all  the 
above-mentioned  carbon  nuclei  exist  in  the  jirotein  molecule.  It  is  not 
inconceivable  that  in  the  hydrolysis  certain  carbon  nuclei  may  be 
secondarily  formed  from  others.  We  cannot  exclude  the  possibility,  as 
suggested  by  Loew,'  that  in  the  hydrolysis  a  marked  atomic  displacement 
perhaps  occurs  before  cleavage,  and  for  this  reason  two  carbon  nuclei, 
such  as  leucine  and  lysine,  or  tyrosine  and  phenylalanine,  may  be  produced 
from  the  same  atomic  groupings,  each  according  to  the  nature  of  the  neigh- 
boring groups. 

Even  if  we  admit  the  above,  still  it  is  undoubtedly  true  that  the  chief 
cleavage  products  of  the  protein  substances  are  amino-acids.  Emil  Fischer 
has  shown  that  the  amino-acids  have  the  property  of  readily  grouping 
together  when  water  is  split  off  and  the  amide  group  of  one  amino-acid 
unites  with  the  carboxyl  group  of  the  other.  In  accord  with  this  behavior 
w'e  can,  as  Hofmeister  2  has  explained,  consider  the  proteins  as  chiefly 
formed  by  the  condensation  of  amino-acids,  where  the  amino-acids  are  united 
to  each  other  by  means  of  imino-groups  according  to  the  following  scheme: 

— NH.CH.CO— NH.CH.CO NH.CH.CO— NH.CH.CO— 

I  I  I  I 

C4H9  CH2.C6H4(OH)  CH2.COOH    C3H6.CH2.NH2 

(Leucine)  (Tyrosine)  (Aspartic  acid)  (Lysine) 

Closely  connected  with  this  conception  is  the  question  whether  it  is 
possible  to  prepare  protein-like  substances  synthetically.  In  this  con- 
nection we  must  mention  that  Grimaux  and  later  also  Schijtzenberger 
and  Pickering  have  been  able  to  prepare  substances  which  in  many  prop- 
erties are  similar  to  the  proteins,  from  various  amino-acids  either  alone  or 
mixed  with  other  bodies  such  as  biuret,  alloxan,  xanthine,  or  ammonia. 
Of  special  interest  are  the  investigations  of  Curtius  and  his  collaborators, 

'  Loew,  Die  chem.  Energie  d.  lebenden  Zellen,  Miinchen,  1898,  and  Hofmeister's 
Beitrago,   1. 

^"Uber  den  Bau  des  Eiweissmolekiils."  Gesellsch.  deutsch.  Naturforscher  and 
Artze,  Verhandl.  1902,  and  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1,  759. 


POLYPEPTIDES.  35 

in  which  they  were  able  to  prepare  synthetically  the  so-called  biuret  base 
(triglycyl-glycine  ethyl  ester)  and  subsequently  many  other  bodies  which 
were  related  to  the  proteins.  The  most  important  work  on  the  chaining  of 
amino-acids  has  been  performed  by  E.  Fischer  ^  and  his  pupils.  They  hav& 
prepared  a  large  number  of  complex  bodies  called  polypeptides,  which, 
according  to  whether  they  contain  two  or  more  amino-acid  groups  united 
together,  are  called  di-,  tri-,  tetrapeptides.  etc.  As  examples  of  polypeptides 
we  ^ill  mention — dipeptides:  glycylalanine.  leucyW-tyrosine,  propylalanine, 
diaminopropionic-acid  dipeptide,  lysyl-lysine,  liistidyl-histidine;  tripep- 
tides:  diglycyl-glycine,  leucyl-alanyl-glycine,  dileucyl cystine;  tetrapeptides: 
triglycyl-glycine,  dileucy  1-glycyl-glycine ;  penta}>eptide :  tetraglycyl-glycine. 

In  connection  with  these  syntheses  it  is  important  to  note  that  E. 
Fischer  and  Bergell,^  by  the  decomposition  of  a  protein  substance, 
fibroin, by  successive  action  of  acid,  proteolytic  enzyme  (tr}-psin),  andbaryta- 
water,  were  able  to  obtain  a  dipeptide.  probably  glycylalanine.  Levexe 
and  Beatty  have  obtained  a  dipeptide  anhydride,  prolineglycyl  anhydride, 
in  the  tryptic  digestion  of  gelatine,  and  Fischer  and  Abderhaldex  ^  have 
also  isolated  from  silk  fibroin  a  dipeptide  composed  of  glycocoU  and  l- 
tyrosine.  In  the  hydrolysis  of  elastin  with  sulphuric  acid  they  obtained  a 
third  dipeptide,  which,  like  the  others,  was  an  anhydride,  namely,  glycyl-Z- 
leucine  anhydride.  Of  the  synthetically  prepared  polypeptides  several  give 
the  biuret  reaction,  and  in  this  regard,  as  well  as  their  behavior  towards 
other  reagents,  they  are  similar  to  the  peptones,  which  will  be  discussed  later- 
Certain  polypeptides,  like  the  biuret  base  'according  to  Schwarzschild)^ 
the  glycyl-Z-tyrosine,  and  alanyl-glycine,  are  split  by  trypsin,  while  others, 
like  glycyl-glycine  and  glycylalanine,  are  not  attacked  by  thii  me  (see 

Chapter  IX). 

It  is  admitted  that  the  atomic  chaining  in  the  protein  consists  of  a 
union  of  a-amino-acids  by  means  of  the  imide  bonds.  It  is  probable  that 
also  other  Unkings  occur,  and  besides  the  above-mentioned  bondage  we 
certainly  have  one  other,  namel}',  the  urea-forming  group  (the  guanidine 
residue)  united  by  the  imide  Unkings  \\'ith  the  ornithine  (diaminovalerianic 
acid).     This  imide  linlvage  is  not  ruptured,  like  that  of  the  a-amino-acids,. 


'  See  Pickering,  Kiag's  College,  London,  Physiol.  Lab.  Collect.  Papers,  1897,  whichr 
also  cites  Grimaux's  work;  also  Joum.  of  Physiol.,  18,  and  Proceed.  Roy.  Soc,  60,, 
1897;  Schiitzenberger,  Compt.  rend.,  106  and  112;  Curtius,  Joum.  f.  prakt.  Chem.. 
(X.  F.),  26  and  70,  and  Ber.  d.  d.  chem.  Gesellsch..  37;  Fischer  and  collaborators, 
ibuL,  35, 36, 37,  38,  39,  and  .\nnal.  d.  Chem.  u.  Pharm.,  340.  AU  the  work  of  E.  Fischer 
and  his  collaborators  on  this  subject  may  be  found  in  E.  Fischer's  Untersuchimgen 
iiber  Aminosauren,  Polj-peptide  and  Proteine  (1899-1906),  Berlin,  1906. 

2  See  Biochem.  Centralbl.,  1,  p.  84. 

'  Levene  and  Beatty,  Ber.  d.  d.  chem.  Gesellsch.,  39,  p.  20G0;  Fischer  and 
Abderhalden,  ibid.,  39,  p.  2315. 


"36  THE  PROTEIN  SUBSTANCES. 

by  trypsin,  but  it  is  by  another  enzyme  discovered  by  Kossel  and  Dakix/ 
called  arginase. 

If  we  consider  the  proteins  as  composed  chiefly  of  amino-acids  combined 
together  in  imide-like  complexes  containing  also  several  NH2  groups  at  the 
ends  of  the  chains,  it  is  easy  to  understand  that  the  proteins,  like  the  amino- 
Acids,  are  amphoteric  electrolytes,  combining  with  bases  as  well  as  with 
acids  to  form  salts  which  are  strongly  dissociated  hydrolytically.  As  we 
must  also  admit  of  the  presence  in  the  protein  molecule  of  a  large  number 
of  COOH  as  well  as  XH2  groups,  it  follows  that  the  protein  bodies  may  be 
polybasic  acids,  as  well  as  polyacidic  bases.  In  this  regard  the  various 
proteins  behave  somewhat  differently,  as  some  of  them,  like  the  protamines, 
are  strongly  basic,  while  others,  like  casein,  behave  chiefly  like  acids,  while 
others  take  a  certain  intermediate  position.  On  this  behavior  as  well  as  on 
their  chemical  constitution  it  is  unfortunately  impossible  to  base  a  proper 
classification  of  the  protein  substances.  Their  general  properties,  such  as 
solubilities  and  precipitation  properties,  are  too  uncertain  to  aid  us  in  the 
construction  of  a  proper  classification.  On  the  other  hand  a  classification 
is  important,  and  we  cannot  do  without  one,  so  we  will  give  the  following 
systematic  summary  of  the  chief  groups  of  the  protein  bodies  as  suggested 
by  Hoppe-Seyler  and  Drechsel,  which  will  be  of  some  aid  to  us. 

I.  Simple  Proteids  or  Albuminous  Bodies. 

,  I  Seralbumin, 

(  Lactalbumin,  and  others.  ) 
r  Fibrinogen, 

-Globulins -<  Myosin, 

f  Serglohvlins,  and  others.  ) 

Nucleoalbumins -I  ^       .  '  .  ,     ^, 

( Ovovitellin,  and  others. 

.„        .     ^  (  Acid  albuminate, 

Albuminates 1    ,  i?    7  ■     71,       •     ^ 

I  Alkali  albuminate. 

Proteoses  (and  Peptones). 

C  1  t  d  P    t  'd  i  Fibrin, 

^  I  Proteids  coagulated  by  heat,  and  others. 

Histones  (Protamines). 

II.  Compound  Proteids. 
Haemoglobins. 

!  Mucins  and  Mncinoids, 
Amyloid, 
Ichthidin,  and  others. 

_,     ,  ^  .  -  ( Nucleohi stone, 

Nucleoproteids -^  „  ,    ,  ,  .  ,     , , 

( Cytoglobin,  and  others. 

*  Zeitschr.  f.  physiol.  Chem.,  41. 


SIMPLE  PROTEIDS.  37 

III.  Albumoids  or  Albuminoids. 

Keratins. 

Elastin. 
Collagen. 
Reticulin. 
(Fibroin,  Sericin,  Comein,  Spongtn,  Conchiolin,  Byssus,  and  others.) 

To  this  summary  must  be  added  that  we  often  find  in  the  investigation? 
of  animal  fluids  and  tissues  protein  substances  which  do  not  fall  in  with 
the  above  scheme,  or  are  classified  only  with  difficulty.  At  the  same  time 
it  must  be  remarked  that  bodies  will  be  found  which  seem  to  rank  between 
the  different  groups,  hence  it  is  very  difficult  to  sharply  divide  these  groups. 

I.  Simple  Proteids  or  Albuminous  Bodies. 

The  simple  proteids  are  never-failing  constituents  of  the  animal  and 
vegetable  organisms.  They  are  especially  found  in  the  animal  bod}- ,  where 
they  form  the  solid  constituents  of  the  muscles  and  of  the  blood-serum,  and 
they  are  so  generally  distributed  that  there  are  onl}-  a  few  animal  secre- 
tions and  excretions,  such  as  the  tears,  the  perspiration,  and  perhaps  the 
urine,  in  which  the}'  are  entirely  absent  or  occur  only  in  traces. 

All  proteids  contain  carbon,  hydrogen,  nitrogen,  oxygen,  and  sulphur;'^ 
a  few  contain  also  phosphorus.  Iron  is  generally  found  in  traces  in  their 
ash,  and  it  seems  to  be  a  regular  constituent  of  a  certain  group  of  the 
albuminous  bodies,  namely,  the  nucleoalbumins.  The  composition  of 
the  different  albuminous  bodies  varies  a  little,  but  the  variations  are  within 
relatively  close  limits.  For  the  better-studied  animal  proteids  the  follow- 
ing composition  of  the  ash-free  substance  has  been  found: 

C 50 . 6  — 54 . 5    per  cent. 

H 6.5  —  7.3 

N 15.0  —17.6 

S 0.3  —  2.2 

P 0.42—  0.85 

0 21.50—23.50 

The  animal  proteids  are  odorless,  tasteless,  and  ordinarily  amorphous. 
The  crystalloid  spherules  {D otter pldttchen)  occurring  in  the  eggs  of  certain, 
fishes  and  amphibians  do  not  consist  of  pure  proteids,  but  of  proteids 
containing  large  amounts  of  lecithin,  which  seem  to  be  combined  with 

'  See  foot-note  l,p.28. 


S8  THE  PROTEIN  SUBSTANCES. 

mineral  substances.  Crystalline  proteids  ^  have  been  prepared  from  the 
seeds  of  various  plants,  and  crystallized  animal  proteids  (see  seralbumin 
and  ovalbumin,  Chapters  YI  and  XIII)  can  be  readily  prepared.  In  the  dry 
condition  the  proteids  appear  as  white  powders,  or  when  in  thin  layers 
as  yellowish,  hard,  transparent  plates.  A  few  are  soluble  in  water,  others 
only  soluble  in  salt  or  faintly  alkaline  or  acid  solutions,  while  others  are 
insoluble  in  these  solvents.  Solutions  of  proteids  are  optically  active  and 
turn  the  plane  of  polarized  light  to  the  left.  All  proteids  when  burned 
leave  an  ash,  and  it  is  therefore  questionable  whether  there  exists  any 
proteid  body  which  is  soluble  in  water  without  the  aid  of  mineral  sub- 
stances. Nevertheless  it  has  not  been  thus  far  successfully  proved  that 
a  native  proteid  body  can  be  prepared  perfectly  free  from  mineral  sub- 
stances without  changing  its  constitution  or  its  properties.^ 

As  previously  stated,  the  albuminous  bodies  are  amphoteric  electrolytes 
and  are  poly  acidic  bases  as  well  as  poly  basic  acids.  The  base-  and  acid- 
combining  powers  of  various  proteids  are  different,  and  the  maximum  acid- 
combining  power  may  perhaps  also  be  used  in  the  differentiation  of  the 
various  proteids  (Cohnheim,  Erb,  and  others). 

The  acid-combining  power  of  the  proteids  has  been  studied  by  means  of 
physical  methods  by  Sjoquist,  Bugarsky,  and  Liebermann  and  with  the  aid 
of  chemical  methods  by  Spiro  and  Pemsel,  Erb,  Cohnheim  and  Krieger, 
V.  Rhorer.  The  methods  pursued  by  Cohnheim  and  Krieger  consisted  in 
precipitating  the  proteid  from  acid  solution  (HCl)  with  an  alkaloid  reagent 
(calcium  phosphotungstate).  The  reaction  took  place  as  follows:  proteid 
hydrochloride  +  calcium  phosphotungstate  =  proteid  phosphotungstate  +  calcium 
chloride.  The  acid  remaining  in  the  filtrate  was  determined,  and  when  this 
quantity  was  subtracted  from  the  known  original  amount  in  the  proteid  solu- 
tion, the  difference  represented  the  acid  combined  with  the  proteid.  If  sodium 
picrate  or  potassium-mercuric  iodide  is  used  instead  of  the  phosphotungstate 
we  have,  according  to  v.  Rhorer,^  a  method  which  is  the  best  of  all  heretofore 
suggested. 

The  proteids  can  be  salted  out  from  their  neutral  solutions  by  neutral 
salts  (NaCl,  Na2S04,  MgS04,  (NH4)2S04,  and  many  others)  in  sufficient 
■concentrations.  While  by  other  precipitants  they  are  often  changed  or 
modified,  their  properties  remain  unchanged  on  salting  out,  and  the  process 
is  reversible,  as  on  diminishing  the  concentration  of  the  salt  the  precipitate 
redissolves.      Spiro  •*  has  shown  that  we  are  not  dealing  here  with  the 


'  See  Maschke,  Journ.  f.  prakt.  Chem.,  74;  Drechsel,  ibid.  (N.  F.),  19;  Griibler, 
ibid.  (N.  F.),  23;  Ritthausen,  ihul.  (N.  F.),  25;  Schmiedeberg,  Zeitschr.  f.  physiol. 
Chem.,  1;  Weyl,  ibid.,  1. 

^  See  E.  Hamack,  Ber.  d.  d.  chem.  Gesellsch.,  22,  23,  25,  and  31;  Werigo,  Pfliiger's 
Archiv,  48;  Billow,  ibid.,  58;   Schulz,  Die  Grosse  des  Eiweissmolokiils,  Jena,  1903. 

^  Pfliiger's  Arch.,  90.  In  regard  to  the  literature  on  this  subject  see  Cohnheim, 
Chemie  der  Eivveisskorper,  2.  Aufl.,  pp.  107-109. 

*  Hofmeister's  Beitrage,  4. 


PROPERTIES  OF   THE  PROTEIDS.  39 

formation  of  a  proteid  combination,  but  rather  that  this  is  an  instance  of 
a  division  of  a  body  between  two  solvents.  The  various  proteids  act  in  an 
essentially  different  manner  towards  the  same  salt,  and  also  for  one  and 
the  same  proteid  the  behavior  towards  different  neutral  salts  is  different, 
as  some  cause  a  precipitate,  while  others  on  the  contrary  do  not 
]^recipitate. 

According  to  Pauli  ^  this  can  be  explained  by  the  fact  that  we  have  to  do 
■with  ion  action  and  that  the  precipitation  action  is  the  algebraic  sum  ot  the  antago- 
nistic properties.  If  we  ascribe  the  precipitating  action  to  the  cations  and  a 
retarding  action  upon  precipitation  to  the  anions,  then,  according  as  a  salt  has 
the  positive  cations  or  the  negative  anions  in  excess,  we  obtain  a  precipitation 
action  or  not,  that  is,  it  is  accelerated  or  retarded. 

The  behavior  of  various  proteids  with  one  and  the  same  salt,  su^h  as 
M,!;S04  or  (NH4)2S04,  is  often  made  use  of  in  the  isolation  of  the  proteid, 
and  special  methods  of  separation  are  based  upon  fractional  precipitation. 
Haslam^  has  recently  shown  that  these  methods  may  lead  to  great  errors 
and  give  good  results  only  under  special  conditions. 

The  conditions  are  different  from  those  of  salting  out,  when  the  proteid 
solution  is  precipitated  by  salts  of  the  heavy  metals.  Here  the  precipi- 
tates (often  called  metallic  albuminates)  are  not  true  combinations  in  con- 
stant proportions,  but  are  rather  to  be  considered  as  loose  absorption 
compounds  of  the  proteid  with  the  salt.^  These  reactions  are  irreversible 
ir.  so  far  that  dilution  with  water  or  removal  of  the  salt  by  means  of  dialysis 
does  not  restore  the  unchanged  proteid.  On  the  other  hand  the  precipi- 
tate, at  least  in  certain  cases,  may  be  redissolved  in  an  excess  of  the  salt 
solution  or  of  the  proteid  solution,  and  in  this  sense  the  process  is  a  re- 
versible one. 

The  precipitation  of  proteids  by  salts  stands  in  close  relationship  to  their 
colloidal  nature.  The  protein  bodies  do  not  as  a  rule  diffuse  through  animal 
membranes,  or  only  to  a  very  slight  extent,  and  hence  have  in  most  cases 
a  pronounced  colloidal  nature  in  Graham's  sense.  Certain  of  them, 
especially  the  peptones  and  a  few  proteoses,  which  will  be  discussed  later, 
seem  to  occupy  an  intermediate  position  between  colloids  and  crystalloids, 
as  their  solutions  are  characterized  by  a  lesser  viscosity  and  greater  dif- 
fusibility,  are  not  readily  precipitable  by  alcohol,  not  coagulable  by  heat, 
and  only  slightly  precipitable  by  salts. 

The  solutions  (or  suspensions)  of  proteids  in  water,  the  proteid  hydrosols, 
are  converted  by  various  means  into  proteid  hydrogels.     Of  these  means 

'  Hofmeister's  Beitriige,  3. 

'See  Cohnheim,  Chemie  der  Eiweisskorper,  2.  Aufl.,  1904,  pp.  144-148;  Pinkus, 
Journ.  of  Physiol.,  27;  Pauli,  Hofmeister's  Beitrage,  3,  p.  225;  Haslam,  Joum.  of 
Physiol.,  32. 

'  See  Galeotti,  Zeitschr.  f.  physiol.  Chem.,  40,  42,  and  44. 


40  THE  PROTEIN  SUBSTANCES. 

we  must  specially  mention  the  following:  flocking  out  with  salts,  precipi- 
tation with  alcohol,  and  coagulation  by  means  of  heat. 

The  precipitation  with  alcohol  is  a  reversible  reaction,  as  the  precipitate 
redissolves  on  subsequent  dilution  with  water.  The  proteids  are  changed 
by  the  action  of  alcohol,  some  readily  and  quickly,  others  with  difficulty 
and  very  slowly;  the  proteid  then  becomes  insoluble  in  water  and  is 
modified. 

Those  proteids  which  occur,  according  to  the  common  views,  preformed 
in  the  animal  fluids  and  tissues  and  which  have  been  isolated  from  these  by 
indifferent  chemical  means  without  losing  their  original  properties  are 
called  native  -proteids.  New  modifications  having  other  properties  can 
be  obtained  from  the  native  proteids  by  heating,  by  the  action  of  various 
chemical  reagents  such  as  acids,  alkalies,  alcohol,  and  others,  as  well  as  by 
proteolytic  enzymes.  These  new  proteids  are  called  modified  {"  denatu- 
rierte  ")  proteids,  to  differentiate  them  from  the  native  proteids.  In  the 
scheme  given  on  page  36  the  albumins,  globulins,  and  nucleoalbumins 
belong  to  the  native  proteids,  and  the  acid  or  alkali  albuminates,  the  pro- 
teoses, the  peptones,  and  the  coagulated  proteids  to  the  modified  proteids. 

On  heating  a  solution  of  a  native  proteid  it  is  modified  at  a  different 
temperature  for  each  different  proteid.  With  proper  reaction  and  other 
favorable  conditions,  for  instance  in  the  presence  of  neutral  salts,  most 
proteids  can  in  this  w^ay  be  precipitated  in  a  solid  form  as  coagulated  pro- 
teid. The  hydrosol  is  converted  into  hydrogel,  but  as  a  modification 
takes  place,  this  process  is  irreversible.  The  various  temperatures  at  which 
coagulation  of  different  proteids  occurs  in  neutral  solutions  containing  salt 
have  in  many  cases  given  us  good  means  for  detecting  and  separating 
proteids.  The  views  in  regard  to  the  use  of  these  means  are  somewhat 
divided.^ 

A  modification  can  be  brought  about  also  by  the  action  of  acids,  alka- 
lies, or  salts  of  the  heavy  metals,  in  certain  cases  by  water  alone,  also  by 
the  action  of  alcohol,  chloroform,^  and  ether,  by  violent  shaking,  etc. 

As  colloids  3  the  proteids  can,  like  other  protein  substances,  to  a  more 


*  See  Halliburton,  Joum.  of  Physiol.,  5  and  11;  Corin  and  Berard,  Bull,  de  I'Acad. 
roy.  de  Belg.,  15;  Haycraft  and  Duggan,  Brit.  Med.  Joum.,  1890,  and  Proc.  Roy. 
Soc.  Edin.,  1889;  Corin  and  Ansiaux,  Bull,  de  I'Acad.  roy.  de  Belg.,  21;  L.  Fredericq, 
Centralbl.  f.  Physiol.,  3;  Haycraft,  ibid.,  4;  Hewlett,  Joum.  of  Physiol.,  13;  Duclaux, 
Annal.  Institut  Pasteur,  7.  In  regard  to  the  relationship  of  the  neutral  salts  to  the 
heat  coagulation  of  albumins  see  also  Starke,  Sitzungsber.  d.  Gesellsch.  f.  Morph.  u. 
Physiol,  in  Miinchen,  1897;   PauH,  Pfliiger's  Arch.,  78. 

2  See  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  31;  Fr.  Kriiger,  Zeitschr.  f.  Biologie, 
41;   Loew  and  Aso,  Bull.  Coll.  Agric.  Tokio,  4. 

'The  study  of  colloids  and  especially  their  changes  of  state  is  of  the  greatest 
importance  for  the  chemistry  of  the  proteids  as  well  as  for  biochemistry  in  general. 
As  the  views  on  important  points  in  this  extensive  subject  are  so  very  divergent,  it  is 


PRECIPITATION   REACTIONS.  41 

or  less  degree,  prevent  the  precipitation  of  a  colloidal  metallic  solution 
(gold  solution)  by  means  of  an  electrolyte  (see  gold  equivalent  according 
to  ZsiGMoxDY  and  Schulz).^ 

The  general  reactions  for  the  proteids  are  very  numerous,  but  only  the 
most  important  will  be  given  here.  To  facilitate  the  study  of  these  they 
have  been  divided  into  the  two  following  groups: 

A.    Precipitation  Reactions  of  the  Proteid  Bodies. 

1.  Coagulation  Test.  An  alkaline  proteid  solution  does  not  coagulate 
on  boiling,  a  neutral  solution  only  partly  and  incompletely,  and  the  reaction 
must  therefore  be  acid  for  coagulation.  The  neutral  liquid  is  first  boiled 
and  then  the  proper  amount  of  acid  added  carefull}'.  A  flocculent  precipi- 
tate is  formed,  and  if  properly  done  the  filtrate  should  be  water-clear.  If 
dilute  acetic  acid  be  used  for  this  test,  the  liquid  must  first  be  boiled  and 
then  1,  2,  or  3  drops  of  acid  added  to  each  10-15  c.c,  depending  on  the 
amount  of  proteid  present,  and  boiled  before  the  addition  of  each  drop.  If 
dilute  nitric  acid  be  used,  then  to  10-15  c.c.  of  the  previously  boiled  liquid 
15-20  drops  of  the  acid  must  be  added.  If  too  little  nitric  acid  be  added,  a 
soluble  combination  of  the  acid  and  proteid  is  formed,  which  is  precipitated 
by  more  acid.  A  proteid  solution  containing  a  small  amount  of  salts 
must  first  be  treated  with  about  1  per  cent  NaCl,  since  the  heating  test 
may  fail,  especially  on  using  acetic  acid,  in  the  presence  of  only  a  slight 
amount  of  proteid.  2.  Behavior  towards  Mineral  Acids  at  Ordinal^  Tem- 
peratures. The  proteids  are  precipitated  by  the  three  ordinary  mineral 
acids  and  by  metaphosphoric  acid,  but  not  by  orthophosphoric  acid.  If 
nitric  acid  be  placed  in  a  test-tube  and  the  proteid  solution  be  allowed  to 
flow  gently  thereon,  a  white  opaque  ring  of  precipitated  proteid  will  form 
where  the  two  liquids  meet  (Heller's  albumin  test).  3.  Precipitation  by 
Metallic  Salts.  Copper  sulphate,  neutral  and  basic  lead  acetate  (in  small 
amounts),  mercuric  chloride,  and  other  salts  precipitate  proteid.  On  this 
is  based  the  use  of  proteids  as  antidotes  in  poisoning  by  metallic  salts.  4. 
Precipitation  by  Ferro-  or  Ferricyanide  of  Potassium  in  Acetic-acid  Solution, 
In  these  tests  the  relative  quantities  of  reagent,  proteid,  or  acid  do  not 
interfere  with  the  delicacy  of  the  test.  5.  Precipitation  by  Neutral  Salts,  such 
as  Na2S04  or  NaCl,  when  added  to  saturation  to  the  liquid  acidified  with 
acetic  acid  or  hydrochloric  acid.  6.  Precipitation  by  Alcohol.  The  solutioa 
must  not  be  alkaline,  but  must  be  either  neutral  or  faintly  acid.     It  must^ 


impossible  to  give  a  short  review  of  the  subject,  hence  we  can  only  refer  for  the  litera- 
ture to  Hamburger,  Osmotischer  Druck  und  lonenlehre  in  den  med.  Wissenschaften, 
Wiesbaden,  1902-1904;  H.  Aron,  Uber  organische  Kolloide,  Biochem.  Centralbl.,  3» 
pp.  461   and   .501. 

'  Hofmeister's  Beitrage,  3. 


42  THE  PROTEIN  SUBSTANCES. 

at  the  same  time,  contain  a  sufficient  quantity  of  neutral  salts.  7.  Precipi- 
tation by  Tannic  Acid  in  acetic-acid  solutions.  The  absence  of  neutral 
salts  or  the  presence  of  free  mineral  acids  may  prevent  the  appearance  of 
the  precipitate,  but  after  the  addition  of  a  sufficient  quantity  of  sodium 
acetate  the  precipitate  will  in  both  cases  appear.  8.  Precipitation  by 
Phosphotungstic  or  Phosphomolybdic  Acids  in  the  presence  of  free  mineral 
acids.  Potassium-mercuric  iodide  and  potassium-bismuth  iodide  precipitate 
proteid  solutions  acidified  with  hydrochloric  acid.  9.  Precipitation  by 
Picric  Acid  in  solutions  acidified  by  organic  acids.  10.  Precipitation  by 
Trichloracetic  Acid  in  2-5  per  cent  solutions.  11.  Precipitation  by  Sali- 
cylsulphonic  Acid.  The  proteids  are  precipitated  by  nucleic  acids,  tauro- 
cholic  and  chondroit in-sulphuric  acid  in  acid  solutions. 

B.    Color  Reactions  for  Proteid  Bodies. 

1.  Millon's  Reaction.^  A  solution  of  mercury  in  nitric  acid  containing 
some  nitrous  acid  gives  a  precipitate  with  proteid  solutions  which  at  the 
ordinary  temperature  is  slowly,  but  at  the  boiling-point  more  quickly, 
colored  red;  and  the  solution  may  also  be  colored  a  feeble  or  bright  red. 
Solid  albuminous  bodies,  when  treated  by  this  reagent,  give  the  same 
coloration.  This  reaction,  which  depends  on  the  presence  of  the  aromatic 
group  in  the  proteid,  is  also  given  by  tyrosine  and  other  monohydroxyl 
benzene  derivatives.  According  to  0.  Nasse  2  it  is  best  to  use  a  solution 
of  mercuric  acetate  which  is  treated  with  a  few  drops  of  a  1  per  cent  solu- 
tion of  potassium  or  sodium  nitrite;  previous  to  use  a  few  drops  of  acetic 
acid  are  added.  2.  Xanthoproteic  Reaction.  With  strong  nitric  acid  the 
albuminous  bodies  give,  on  heating  to  boiling,  yellow  flakes  or  a  yellow 
solution.  After  making  alkaline  with  ammonia  or  alkalies  the  color  becomes 
orange-yellow.  3.  Adamkiewicz' s  Reaction.  If  a  little  proteid  is  added 
to  a  mixture  of  1  vol.  concentrated  sulphuric  acid  and  2  vols,  glacial  acetic 
acid  a  reddish-violet  color  is  obtained  slowly  at  ordinary  temperatures,  but 
more  quickly  on  heating.  According  to  Hopkins  and  Cole  ^  this  reaction 
takes  place  only  on  using  glacial  acetic  acid  containing  glyoxylic  acid. 
According  to  them  it  is  better  to  use  a  solution  of  glyoxylic  acid,  which  can 
be  readily  prepared  b}'  adding  sodfum  amalgam  to  a  concentrated  solution 
of  oxalic  acid  and  filtering  after  the  discharge  of  the  gas.   •  A  dilute  aqueous 


'  The  reagent  is  obtained  in  the  following  way:  1  pt.  mercury  is  dissolved  in  2  pts. 
nitric  acid  (of  sp.  gr.  1.42),  first  cold  and  then  warmed.  After  complete  solution  of 
the  mercury  add  1  volume  of  the  solution  to  2  volumes  of  water.  Allow  this  to  stand 
a  few  hours  and  decant  the  supernatant  liquid. 

'  See  O.  Nasse,  Sitzungsb.  d.  Naturforsch.  Gesellsch.  zu  Halle,  1879,  and  Pfliiger'a 
Arch.,  S3;   see  also  Vaubel  and  Blum,  Joum.  f.  prakt.  Chem.  (N.  F.),  57. 

*  Proceed.  Roy.  Soc,  68, 


COLOR  REACTIONS.  43 

solution  of  the  acid  or  some  of  the  soHd  acid  is  added  to  the  proteid  s6lu- 
tion  and  sulphuric  acid  allowed  to  flow  do\\Ti  the  side  of  the  test-tube,  when 
the  reddish-violet  color  will  appear  at  the  point  of  contact  of  the  two 
liquids.  Gelatine  does  not  give  this  reaction.  4.  Biuret  test.  If  a  proteid 
solution  be  first  treated  with  caustic  potash  or  soda  and  then  a  dilute  cop- 
per-sulphate solution  be  added  drop  by  drop,  first  a  reddish,  then  a  red- 
dish-violet, and  lastly  a  violet-blue  color  is  obtained.  5.  Proteids  are 
sofuble  on  heating  with  concentrated  hydrochloric  acid,  producing  a  violet 
color,  and  when  they  are  pre\iously  boiled  with  alcohol  and  then  washed 
with  ether  (Liebermaxx  \)  they  give  a  beautiful  blue  solution.  This  blue 
color  is  due,  according  to  Cole,  2  to  a  contamination  of  the  ether  ^^•ith  gly- 
oxylic  acid,  which  reacts  ^^■ith  the  tryptophane  groups  split  off  by  the  hydro- 
chloric acid.  6.  With  concentrated  sulphuric  acid  and  sugar  (in  small  quan- 
tities) the  alljuminous  bodies  give  a  beautiful  red  coloration.  7.  With 
;)-dimethylaminobenzaldehyde  and  concentrated  sulphuric  acid  the  pro- 
teids give  a  beautiful  reddish-\iolet  or  deep-violet  coloration  (0.  Neubauer 
and  E.  Rohde^). 

Many  of  these  color  reactions  are  obtained,  as  shown  by  Salkowski,'  by  the 
aromatic  or  heterocyclic  cleavage  products  of  the  proteids.  Millon's  reaction 
is  given  only  by  the  substances  of  the  phenol  group;  the  xanthoproteic 
reaction  by  the  phenol  group  and  skatol  or  skatolcarbonic  acid.  Liebermaxn's 
reaction  depends,  according  to  Cole,  upon  the  skatol  (indol)  group,  and  the  reac- 
tions with  sulphuric  acid  and  sugar  (Cole)  and  with  dimethylaminobenzaldehyde 
(Rohde)  are  also  caused  by  this  group.  Ada.mkiewicz's  reaction  is  given  only  by 
the  bodies  of  the  indol  group.  The  biuret  reaction  is  not  only  given  by  pro- 
tein substances  but  also  by  many  other  bodies.  According  to  H.  Schiff  ^  this 
reaction  occurs  with  those  bodies  containing  amino  groups,  COXHo,  CSNH,, 
C(XH)XH2  or  also  CHoXH,,  united  either  directly  by  their  carbon  atoms  or  by 
means  of  a  third  carbon  or  nitrogen  atom.  As  examples  of  such  bodies  we  can 
mention  several  diamines  or  aminoamides,  such  as  oxamide,  biuret,  glycinamide, 
a-  and  ,5-aminobutyramide,  aspartic-acid  amide,  etc.,  although  we  are  not  dear 
as  to  the  conditions  necessary  for  the  bringing  about  of  this  reaction.  The  biuret 
reaction  alone  is  therefore  no  proof  as  to  the  protein  nature  of  a  substance — for 
example,  urobilin  gives  a  very  similar  color  reaction — and  a  protein  substance  can 
still  retain  its  protein  nature,  as  by  the  action  of  nitrous  acid  or  by  a  splitting 
off  of  ammonia,  although  it  does  not  give  the  biuret  reaction. 

The  delicacy  of  the  various  reagents  differs  for  the  different  proteids, 
and  on  this  account  it  is  impossible  to  give  the  degree  of  delicacy  for  each 
reaction  for  all  proteids.  Of  the  precipitation  reactions,  Heller's  test  (if 
we  eliminate  the  peptones  and  certain  proteoses)  is  recommended  in  the 
first  place  for  its  delicacy,  though  it  is  not  the  most  delicate  reaction,  and 

'  Centralbl.  f.  d.  med.  Wissensch.,  1887. 

^  Joum.  of  Physiol.,  30. 

'  Zeitschr.  f.  physiol.  Chem.,  44. 

*  Ibid. ,12. 

^  Ber.  d.  d.  chem.  Gesellsch.,  29  and  30. 


44  THE  PROTEIN  SUBSTANCES. 

because  it  can  be  performed  so  easily.  Among  the  precipitation  reactions, 
that  with  basic  lead  acetate  (when  carefully  and  exactly  executed)  and  the 
reactions  6,  7,  8,  9,  and  11  are  the  most  delicate.  The  color  reactions  1  to 
4  show  great  delicacy  in  the  order  in  which  they  are  given. ^ 

No  proteid  reaction  is  in  itself  characteristic,  and,  therefore,  in  testing 
for  proteids  one  reaction  is  not  sufficient,  but  a  number  of  precipitation  and 
color  reactions  must  be  employed. 

For  the  ciuantitative  estimation  of  coagulable  proteids  the  determina- 
tion by  boiling  with  acetic  acid  can  be  performed  with  advantage,  since,  by 
operating  carefully,  it  gives  exact  results.  Treat  the  proteid  solution  with 
a  1-2  per  cent  common-salt  solution,  or  if  the  solution  contains  large  amounts 
of  proteid  dilute  with  the  proper  quantity  of  the  above  salt  solution,  and 
then  carefully  neutralize  with  acetic  acid.  Now  determine  the  quantity 
of  acetic  acid  necessary  to  completely  precipitate  the  proteids  in  small 
measured  portions  o  the  neutralized  liquid  which  have  previously  been 
heated  on  the  water-bath,  so  that  the  filtrate  does  not  respond  to  Hel- 
ler's test.  Now  warm  a  larger  weighed  or  measured  quantity  of  the  liquid 
on  the  water-bath,  and  add  gradually  the  required  quantity  of  acetic 
acid,  with  constant  stirring,  and  continue  heating  for  some  time.  Filter, 
wash  with  water,  extract  with  alcohol  and  then  with  ether,  dry,  weigh, 
incinerate,  and  weigh  again.  With  proper  work  the  filtrate  should  not  give 
Heller's  test.  This  method  serves  in  most  cases,  and  especially  so  in 
cases  where  other  bodies  are  to  be  quantitatively  estimated  in  the  filtrate. 

The  precipitation  by  means  of  alcohol  may  also  be  used  in  the  quan- 
titative estimation  of  proteids.  The  liquid  is  first  carefully  neutralized, 
treated  with  some  NaCl  if  necessary,  and  then  alcohol  added  until  the 
solution  contains  70-80  vol.  per  cent  anhydrous  alcohol.  The  precipitate 
is  collected  on  a  filter  after  24  hours,  extracted  with  alcohol  and  ether, 
dried,  weighed,  incinerated,  and  again  weighed.  This  method  is  only 
applicable  to  liquids  which  do  not  contain  any  other  substances,  like  glyco- 
gen, which  are  insoluble  in  alcohol. 

In  both  of  these  methods  small  quantities  of  proteid  may  remain  in  the 
filtrates.  These  traces  may  be  determined  as  follows:  Concentrate  the 
filtrate  sufficiently,  remove  any  separated  fat  by  shaking  with  ether,  and 
then  precipitate  with  tannic  acid.  Approximately  63  per  cent  of  the  tannic- 
acid  precipitate,  washed  with  cold  water  and  then  dried,  may  be  considered 
as  proteid. 

In  many  cases  good  results  may  be  obtained  by  i^recipitating  all  the 
proteid  with  tannic  acid  and  determining  the  nitrogen  in  the  washed  pre- 
cipitate by  means  of  Kjeldahl's  method.  On  multiplying  the  quantity 
of  nitrogen  found  by  6.25  we  obtain  the  quantity  of  proteid. 

The  removal  of  proteids  from  a  solution  may  in  most  cases  be  performed 
by  boiling  with  acetic  acid.  Small  amounts  of  proteid  which  remain  in  the 
filtrates  may  be  separated  by  boiling  with  freshly  precipitated  lead  car- 
bonate or  with  ferric  acetate,  as  described  by  Hofmeister.^  If  the  liquid 
cannot  be  boiled,  the  proteid  may  be  precipitated  by  the  very  careful  addi- 

'  In  regard  to  the  precipitation  and  coloration  reactions  of  proteids  with  aniline 
dye.s  see  Heidenhain,  Pfliiger's  Arch.,  90,  9G. 
Zeitschr.  f.  physiol.  Chem.,  2  and  4. 


ALBUMINS  AND  GLOBULINS.  45 

tion  of  lead  acetate,  or  by  the  addition  of  alcohol.  If  the  liquid  contains 
substances  which  are  precipitated  by  alcohol,  such  as  glycogen,  then  the 
proteid  may  be  removed  by  the  alternate  addition  of  potassium-mercuric 
iodide  and  hydrochloric  acid  (see  Chapter  VIII,  on  Glycogen  Estimation), 
or  also  by  trichloracetic  acid  as  suggested  by  Obermayer  and  Frankel.^ 

In  the  precipitation  of  proteid  as  well  as  the  quantitative  estin  lation  by  means 
of  heat,  it  must  be  borne  in  mind,  as  shown  by  Spiro,"  that  several  nitrogenous 
substances,  such  as  piperidine,  pyridine,  urea,  etc.,  disturb  the  coigulation  of  the 
proteids. 

Synopsis  of  the  Most  Important  Properties  of  the  Different  Groups  of 

Proteids. 

As  it  is  not  possible  to  base  the  classification  of  the  different  proteid 
groups  according  to  their  constitution,  we  are  obliged  to  make  use  of  their 
different  solubilities  and  precipitation  properties  in  their  general  characteri- 
zation. As  there  exist  no  sharp  differences  between  the  various  groups  in 
this  regard  it  is  impossible  to  draw  a  sharp  line  between  them. 

Albumins.  These  bodies  are  soluble  in  water  and  are  not  precipitated 
by  the  addition  of  a  little  acid  or  alkali.  They  are  precipitated  by  the 
addition  of  large  quantities  of  mineral  acids  or  metallic  salts.  Their  solu- 
tion in  water  coagulates  on  boiling  in  the  presence  of  neutral  salts,  but  a 
weak  saline  solution  does  not.  If  NaCl  or  i\IgS04  is  added  to  saturation 
to  a  neutral  solution  in  water  at  the  normal  temperature  or  at  30°  C.  no 
precipitate  is  formed;  but  if  acetic  acid  is  added  to  this  saturated  solution 
the  albumins  readily  separate.  When  ammonium  sulphate  is  added  in 
substance  to  saturation  to  an  albumin  solution  a  complete  precipitation 
occurs  at  the  ordinary  temperature.  Of  all  the  native  proteids  the  albumins 
are  the  richest  in  sulphur,  containing  from  1.6  per  cent  to  2.2  per  cent. 

Globulins.  These  substances  are  insoluble  in  water,  but  dissolve  in 
dilute  neutral  salt  solutions.  The  globulins  are  precipitated  unchanged 
from  these  solutions  by  sufficient  dilution  with  water,  and  on  heating  they 
coagulate.  The  globulins  dissolve  in  water  on  the  addition  of  very  little 
acid  or  alkali,  and  on  neutralizing  the  solvent  they  precipitate  again.  The 
solution  in  a  minimum  amount  of  alkali  is  precipitated  by  carbon  dioxide, 
Ixit  the  precipitate  may  be  redissolved  by  an  excess  of  the  precipitant. 
The  neutral  solutions  of  the  globulins  containing  salts  are  partly  or  com- 
pletely precipitated  on  saturation  with  NaCl  or  J\IgS04  in  substance  at 
normal  temperatures.  The  globulins  are  completely  precipitated  by  half- 
saturating  with  ammonium  sulphate.  The  globulins  contain  an  average 
amount  of  sulphur  generally  not  below  1  per  cent. 


'  Obermayer,  Wien.  med.  Jahrbiicher,  1888;   Frankel,  Pfliiger's  Arch.,  52  and  55. 
^  Zeitschr.  f.  physiol.  Chem.,  30. 


46  THE  PROTEIN  SUBSTANCES. 

According  to  J.  Starke  '  the  globulins  are  not  soluble  in  dilute  salt  solutions, 
but  form  alkali  proteid  compounds  whose  solubility  in  salts  is  brought  about  by 
an  increase  in  the  free  OH  ions  produced  by  the  salts.  This  view  is  not  tenable 
for  several  globulins  and  seems  in  fact  not  to  be  well  founded. 

That  a  sharp  line  cannot  be  drawn  between  the  allmmins  and  globnlins 
follows  from  the  fact  that  the  albumins  can  be  converted  into  giob- 
iilins.  The  possibility  of  a  conversion  of  ovalbumin  into  globulin  is  based 
upon  the  observations  of  Starke.  That  a  transformation  of  seralbumin 
into  serglobulin  by  the  aid  of  the  weak  action  of  alkali  in  the  warmth,  with 
the  splitting  off  of  sulphur,  can  take  place,  has  been  more  conclusively 
shown  by  ^Ioll^  by  experimenting  with  blood-serum  as  well  as  with  crys- 
talline seralbumin.  According  to  Moll  first  pseudoglobulin  is  formed 
from  the  seralbumin  and  then  euglobulin  (see  Chapter  VI).  The  artificial 
globulins  thus  obtained  had  the  same  sulphur  content  and  properties  as 
the  natural  products. 

It  is  just  as  difficult  to  draw^  a  sharp  line  between  the  globulins  and 
albuminates  as  it  is  betw'een  the  globulins  and  albumins.  Several  globulins 
are  very  readily  changed  by  the  action  of  vers'  little  acid,  as  also  by  standing 
under  water  when  in  a  precipitated  condition,  into  albuminates,  and  then 
become  insoluble  in  neutral  salt  solutions.  Osborxe,^  who  has  closely 
studied  this  property  in  connection  with  edestin  (from  hemp-seed),  considers 
the  globulin,  "globan, "  which  has  been  made  insoluble  in  salt  solution, 
as  an  intermediate  step  in  the  formation  of  the  albuminate  w'hich  is  pro- 
duced by  the  hydrolytic  action  of  the  H  ions  of  water  or  of  the  acid. 

Nucleoalbumins.  This  group  of  phosphorized  proteids  is  found  widely 
distributed  in  both  the  animal  and  vegetable  kingdoms.  The  nucleoalbumins 
behave  like  weak  acids;  they  are  nearly  insoluble  in  water,  but  dissolve 
easih-  with  the  aid  of  a  little  alkali.  The  nucleoalbumins  resemble  certain 
of  the  globulins  and  albuminates  in  solubility  and  precipitation  properties, 
but  differ  from  these  two  groups  by  containing  phosphorus.  They  stand 
close  to  the  nucleoproteids  by  their  content  of  phosphorus,  but  differ  from 
these  in  not  yielding  any  purine  bases  on  cleavage.  It  has  not  j'et  been 
found  possible  to  obtain  from  the  nucleoalbumins  any  proteid-free  pseudo- 
nucleic  acids  corresponding  to  the  nucleic  acids,  but  only  acids  rich  in 
phosphorus,  which  always  give  the  proteid  reactions  (Levene  and  Alsberg, 
Salkowski  "*).     For  this  reason  the  nucleoalbumins  cannot  be  classed  as 


'  Zeitschr.  f.  Biologie,  40  and  42.  In  regard  to  the  various  views  on  this  subject 
see  Wolff  and  Smits,  ibid.,  41;  Osborne,  1.  c;  Hammarsten,  Ergebnisse  der  Physiologie, 
Jahrg.  I,  Abt.  1. 

^  Hofmeister's  Beitrage,  4  and  7. 

'  Zeitschr.  f.  physiol.  Chem.,  33. 

*  Levene  and  Alsberg,  ibi/1.,  31;   Salkowski,  ibid.,  32;   Levene,  ibid.,  32. 


XUCLEOALBUMIXS   AND   LECITHALBL^uXS.  47 

compound  proteids.  In  peptic  digestion  a  proteid  rich  in  phosphorus  can 
be  split  off  from  most  nucleoalbumins,  and  this  has  been  called  para-  or 
pseudonudein.  The  claim  made  by  LiEBERiL\xx  that  the  pseudonuclein 
is  a  combination  of  proteid  with  metaphosphoric  acid  has  been  shown  to 
be  incorrect  by  the  investigations  of  Giertz.^  The  nucleoalbumins  always 
seem  to  contain  some  iron. 

The  separation  of  pseudonuclein  in  peptic  digestion  is  no  doubt  characteristic 
of  the  nucleoalbumin  group,  but  the  non-appearance  of  the  pseudonuclein  precij^i- 
tate  does  not  entirely  exclude  the  presence  of  a  nucleoalbumin.  The  extent  of 
such  a  cleavage  is  dependent  upon  the  intensity  of  the  pepsin  digestion,  the 
degree  of  acidity,  and  the  relationship  between  the  nucleoalbumins  and  the 
digestive  fluids.  The  .separation  of  a  pseudonuclein  may,  as  shown  by  Salkowski, 
not  occur  even  in  the  digestion  of  ordinary  casein,  and  Wroblewski  did  not 
obtain  any  i^seudonuclein  at  all  in  the  digestion  of  the  casein  from  human  milk. 
WiMAX  -  has  also  .shown  in  the  digestion  of  vegetable  nucleoalbumin  that  the 
obtainment  of  considerable  pseudonuclein  or  none  is  de})endent  upon  the  way  in 
which  the  digestion  is  performed.  The  most  essential  characteristic  of  this  group 
of  proteids  is  that  they  contain  phosphorus,  and  that  the  xanthine  bases  are 
absent  in  their  cleavage  products. 

The  nucleoalbimiins  are  often  confoimded  with  nucleoproteids  and 
also  witli  phosphorized  glucoproteids.  From  the  first  class  they  differ  by 
not  yielding  any  xanthine  bodies  when  boiled  with  acids,  and  from  the 
second  group  by  not  yielding  any  reducing  substance  on  the  same  treat- 
ment. 

Lecithalbumins.  In  the  preparation  of  certain  protein  substances 
products  are  often  obtained  containing  lecithin,  and  this  lecithin  can  only 
be  removed  with  difficulty  or  incompletely  by  a  mixture  of  alcohol  and 
ether.  Ovovitellin  (Chapter  XIII)  is  such  a  protein  body  containing 
considerable  lecithin,  and  Hoppe-Seyler  considers  it  a  combination  of 
proteid  and  lecithin.  Similar  substances  occur  in  fish-eggs.  These  last 
lecithalbumins  often  have  the  solubilities  of  the  globulins  and  are  readily 
soluble  in  dilute  salt  solutions.  The  behavior  of  the  nucleoalbumin  of 
the  eggs  of  the  perch  shows  how  easily  this  solubility  may  be  changed.  This 
nucleoalbumin,  which  contains  considerable  amounts  of  lecithin,  is  readily 
soluble  in  dilute  NaCl  solution,  but  at  ordinary  temperatures  it  is  changed 
by  0.1  per  cent  HCl  nearly  instantaneou.sly  and  without  splitting  off 
lecithin,  so  that  it  becomes  insoluble  in  dilute  salt  solutions  (Hamm.\rsten). 
LiEBERMAXx^  has  obtained  proteids  containing  lecithin  as   an  insoluble 


'  Liebermann,  Ber.  d.  deutsch.  chtm.  Gesellsch..  21;  Giertz,  Zeitschr.  f.  physiol. 
Chem..  28. 

-  Salkowski,  Pfliiger's  Arch.,  63;  Wioljlewski,  Beit  rage  zur  Kenntnis  des  Frauen- 
ka?eins,  Inaug.-Diss.  Bern,  1894;   Wiman,  Upsala  Lakaref.  Forh.  (X.  F.),  2. 

'  Hoppe-Seyler,  Med.  chem.  Untersuch.,  1868;  also  Zeitschr.  f.  physiol.  Chem.,  13, 
479;  Hammarsten,  Skand.  Arch.  f.  Physiol.,  1";  Liebermann,  Pfliiger's  Archiv,  50 
and  54. 


48  THE  PROTEIN  SUBSTANCES. 

residue  on  the  peptic  digestion  of  the  mucous  membrane  of  the  stomach, 
liver,  kidneys,  lungs,  and  spleen.  He  considers  them  as  combinations  of 
proteid  and  lecithin  and  calls  them  lecithalbumins.  Further  investigation 
of  these  bodies  is  desirable. 

Alkali  and  Acid  Albuminates.  The  native  proteids  are  modified  by  the 
action  of  sufficiently  strong  acids  or  alkalies.  By  the  action  of  alkalies 
all  native  albuminous  bodies  are  converted,  with  the  elimination  of  nitro- 
gen, or  by  the  action  of  stronger  alkali,  with  the  extraction  of  sulphur  also, 
into  a  new  modification,  called  alkali  albuminate,  whose  specific  rotation 
is  increased  at  the  same  time.  If  caustic  alkali  in  substance  or  in  strong 
solution  be  allowed  to  act  on  a  concentrated  proteid  solution,  such  as 
l)lood-serum  or  egg-albumin,  the  alkali  albuminate  may  be  obtained  as  a 
solid  jelly  which  dissolves  in  water  on  heating,  and  which  is  called  "  Lieber- 
kuhn's  solid  alkali  albuminate."  By  the  action  of  dilute  caustic  alkali 
solutions  on  dilute  proteid  solutions  we  have  alkali  albuminates  formed 
slowly  at  the  ordinary  temperature,  but  more  rapidly  on  heating.  These 
solutions  may  vary  with  the  nature  of  the  proteid  acted  upon,  and  also 
wnth  the  intensity  of  the  action  of  the  alkali,  but  still  they  have  certain 
reactions  in  common. 

If  proteid  is  dissolved  in  an  excess  of  concentrated  hydrochloric  acid,  or 
If  we  digest  a  proteid  solution  acidified  with  1-2  p.  m.  hydrochloric  acid  in 
the  thermostat,  or  digest  the  proteid  for  a  short  time  with  pepsin-hydro- 
■chloric  acid,  we  obtain  new  modifications  of  proteid  which  indeed  may  show 
somewhat  varying  properties,  but  have  certain  reactions  in  common.  These 
modifications,  which  may  be  obtained  in  a  solid  gelatinous  condition  on 
sufficient  concentration,  are  called  acid  albuminates  or  acid  albumins,  and 
sometimes  syntonin,  though  we  prefer  to  apply  the  term  syntonin  to  the 
acid  albuminate,  which  is  obtained  by  extracting  muscles  with  hydrochloric 
acid  of  1  p.  m. 

The  alkali  and  acid  albuminates  have  the  following  reactions  in  com- 
mon: They  are  nearly  insoluble  in  water  and  dilute  common-salt  solu- 
tion (see  page  46),  but  they  dissolve  readily  in  water  on  the  addition  of  a 
very  small  quantity  of  acid  or  alkali.  Such  a  solution  as  nearly  neutral  as 
possible  does  not  coagulate  on  boiling,  but  is  precipitated  at  the  normal  tem- 
perature on  neutralizing  the  solvent  by  an  alkali  or  an  acid.  A  solution  of 
an  alkali  or  acid  albuminate  in  acid  is  easily  precipitated  on  saturating 
with  NaCl,  but  a  solution  in  alkali  is  precipitated  with  difficulty  or  not  at 
all,  according  to  the  amount  of  alkali  it  contains.  IMineral  acids  in  excess 
})recipitate  solutions  of  acid  as  well  as  alkali  albuminates.  The  nearly  neu- 
tral solutions  of  these  bodies  are  also  precipitated  by  many  metallic  salts. 

Notwithstanding  this  agreement  in  the  reactions,  the  acid  and  alkali 
albuminates  are  essentially  different,  for  by  dissolving  an  alkali  albuminate 
in  some  acid  no  acid  albuminate  solution  is  obtained,  nor  is  an  alkali  al- 


ALKALI  AND   ACID   ALBUMINATES.  49 

buminate  formed  on  dissolving  an  acid  albuminate  in  water  by  the  aid  of 
a  little  alkali.  In  the  first  case  we  obtain  a  combination  of  the  alkali 
albuminate  and  the  acid  solul)le  in  water,  and  in  the  other  case  a  soluble 
combination  of  the  acid  albuminate  with  the  alkali  added.  The  chemical 
process  in  the  modification  of  proteids  with  an  acid  is  essentially  different 
from  the  modification  with  an  alkali,  hence  the  ])roducts  are  of  a  different 
kind.  The  alkali  albuminates  are  relatively  strong  acids.  They  may  be 
dissolved  in  water  with  the  aid  of  CaCOg,  with  the  elimination  of  COo, 
which  does  not  occur  with  typical  acid  albuminates,  and  they  show  in 
opposition  to  the  acid  albuminates  also  other  variations  which  stand  in 
connection  with  their  strongly  marked  acid  nature.  Dilute  solutions  of 
alkalies  act  more  energetically  on  proteids  than  do  acids  of  corresponding 
concentration.  In  the  first  case  a  part  of  the  nitrogen,  and  often  also  the 
sulphur,  is  split  off,  and  from  this  property  we  may  obtain  an  alkali  albu- 
minate by  the  action  of  an  alkali  upon  an  acid  albuminate;  but  we  cannot 
obtain  an  acid  albuminate  by  the  obverse  reaction  (K.  MorxerI).  For 
this  reason  the  designation  of  the  modified  proteid  obtained  by  the  action 
of  alkali  or  acid  as  proteiti,  the  combination  of  this  protein  with  alkali 
as  alkali  albuminate,  and  the  combination  with  acid  as  acid  albuminate, 
leads  to  a  misunderstanding  or  to  a  wrong  conception. 

The  preparation  of  the  albuminates  has  been  given  above.  The  cor- 
responding albuminate  obtained  by  the  action  of  alkalies  or  acids  upon  a 
proteid  solution  may  be  precipitated  by  neutralizing  with  acid  or  alkali. 
The  washed  precipitate  is  dissolved  in  water  by  the  aid  of  a  little  alkali  or 
acid,  and  again  precipitated  by  neutralizing  the  solvent.  If  this  precipi- 
tate which  has  been  washed  in  water  is  treated  with  alcohol  and  ether, 
the  albuminate  will  be  obtained  in  a  pure  form. 

In  the  preparation  of  acid  as  well  as  of  alkali  albuminates,  proteoses  and  the 
nearly  related  albuminates  are  formed.  The  "alkali  albumose"  obtained  by 
Maas  ^  belongs  to  this  class.  The  lysalbinic  acid  and  protalbinic  acid  obtained 
by  Paal  ^  from  ovalbumin  are  likewise  alkali  albuminates.  Desaminoalhuminic 
acid  is  an  alkali  albuminate  which  Schmiedeberg  ^  obtained  by  the  action  of  such 
weak  alkali  that  a  part  of  the  nitrogen  was  evolved,  but  the  quantity  of  sulphur 
remained  the  same.  The  proteid  combination  obtained  by  Blum  by  the  action 
of  formol  on  proteid  and  called  by  him  protogen  has  similarities  with  the  alkali 
albuminates  in  regard  to  solubilities  and  precipitation,  but  is  not  identical 
therewith.^ 


'Pfliiger's  Arch.,  17. 

-  Zeitschr.  f.  physiol.  Chem.,  30. 

'  Ber.  d.  d.  chem.  Gescllsch.,  35. 

'  Arch.  f.  exp.  Path.  u.  Pharm.,  39. 

^  Blum,  Zeitschr.  f.  physiol.  Chem.,  22,  The  older  investigations  of  Loew  may 
be  found  in  Maly's  Jahresber.,  1888.  On  the  action  of  formaldehyde  see  also  Benedi- 
centi,  Arch.  f.  (Anat.  u.)  Physiol.,  1897;  S.  Schwartz,  Zeitschr.  f.  physiol.  Chem.,  30; 
Bliss  and  No%y,  Joum.  of  Exper.  Med.,  4. 


50  THE  PROTEIN  SUBSTANCES. 

Proteoses  and  Peptones.  Peptones  were  formerly  designated  as  the 
final  products  of  the  decomposition  of  protein  bodies  by  means  of  pro- 
teolytic enzymes,  in  so  far  as  these  final  products  are  still  true  proteins, 
while  tlie  intermediate  products  produced  m  the  peptonization  of  proteins, 
in  so  far  as  they  are  not  substances  similar  to  albuminates,  were  designated 
as  proteoses  (albumoses,  or  propeptones).  Proteoses  and  peptones  may  also 
l>e  produced  by  the  hydrolytic  decomposition  of  the  proteins  with  acids 
or  alkalies,  and  by  the  putrefaction  of  the  same.  They  may  also  be  formed 
in  very  small  quantities  as  by-products  in  the  investigations  of  animal  fluids 
and  tissues,  and  the  question  as  to  the  extent  to  which  these  exist  pre- 
formed under  physiological  conditions  requires  very  careful  investiga- 
tion. 

Between  the  peptone,  which  represents  the  final  cleavage  product ,  and 
the  proteose,  which  stands  closest  to  the  original  protein,  we  have  undoubt- 
edly a  series  of  intermediate  products.  Under  such  circumstances  it  is  a 
difficult  problem  to  try  to  draw  a  sharp  line  between  the  peptone  and 
the  proteose  group,  and  it  is  just  as  difficult  to  define  our  conception  of 
peptones  and  proteoses  in  an  exact  and  satisfactory  manner. 

The  proteoses  (or  albumoses)  used  to  be  considered  as  those  protein  bodies 
whose  neutral  or  faintly  acid  solutions  do  not  coagulate  on  boiling,  and 
which,  to  distinguish  them  from  peptones,  were  characterized  chiefly  by 
the  following  properties:  The  watery  solutions  are  precipitated  at  the 
ordinary  temperature  by  nitric  acid,  as  well  as  by  acetic  acid  and  potassium 
ferrocyanide,  and  this  precipitate  has  the  peculiarity  of  disappearing  on 
heating  and  reappearing  on  cooling.  If  a  proteose  solution  is  saturated 
with  NaCl  in  substance,  the  proteose  is  partly  precipitated  in  neutral 
solutions,  but  on  the  addition  of  acid  saturated  with  salt  it  is  more  com- 
pletely precipitated.  This  precipitate,  which  dissolves  on  warming,  is 
a  combination  of  the  proteose  with  the  acid. 

We  formerly  designated  as  peptones  those  protein  bodies  which  are 
readil}'  soluble  in  water  and  which  do  not  coagulate  by  heat,  whose  solutions 
are  precipitated  neither  by  nitric  acid,  nor  by  acetic  acid  and  potassium 
ferrocyanide,  nor  by  neutral  salts  and  acid. 

The  reactions  and  properties  which  the  proteoses  and  peptones  have  in 
common  were  formerly  considered  as  the  following:  They  give  all  the  color 
reactions  of  the  proteins,  but  with  the  biuret  test  they  give  a  more  beautiful 
red  color  than  the  ordinary  proteids.  They  are  precipitated  by  ammoniacal 
lead  acetate,  by  mercuric  chloride,  tannic,  phosphotungstic,  and  phospho- 
molybdic  acids,  by  potassium-mercuric  iodide  and  hydrochloric  acid,  and 
also  by  picric  acid.  They  are  precipitated  but  not  coagulated  by  alcohol, 
that  is,  the  precipitate  obtained  is  solul^le  in  water  even  after  being  in 
contact  with  alcohol  for  a  long  time.  The  proteoses  and  peptones  also 
have  a  greater  diffusive  power  than  native  proteins,  and  the  diffusive 


PROTEOSES  AXD  PEPTONES.  51 

power  is  greater  the  nearer  the  questionable  substance  stands  to  the  final 
product,  the  now  so-called  true  peptone. 

These  old  views  have  gradually  undergone  an  essential  change.  After 
Heyxsius'  1  observation  that  ammonium  sulphate  was  a  general  precipi- 
tant for  proteins,  and  for  peptones  in  the  old  sense,  Kuhxe  and  his  pupils  ^ 
proposed  this  salt  as  a  means  of  separating  proteoses  and  peptones.  Those 
products  of  digestion  which  separate  on  saturating  their  solution  \\ith 
ammonium  sulphate,  or  can  indeed  be  salted  out  at  all,  are  considered 
by  Kuhxe  and  also  by  most  of  the  modern  investigators  as  proteoses,  while 
those  which  remain  in  solution  are  called  peptones  or  true  peptones.  These 
true  peptones  are  formed  in  relativeh'  large  amounts  in  pancreatic  diges- 
tion, while  in  pepsin  digestion  they  are  formed  only  in  small  quantities 
or  after  prolonged  digestion. 

According  to  Schutzexberger  and  Kuhxe  ^  the  proteins  yield  two 
chief  groups  of  new  protein  bodies  when  decomposed  by  dilute  mineral 
acids  or  with  proteolytic  enzymes;  of  these  the  anti  group  shows  a  greater 
resistance  to  further  action  of  the  acid  and  enzyme  than  the  other,  nameh% 
the  hemi  grouj).  These  two  groups  are,  according  to  Kuhxe,  united  in 
the  different  proteoses,  even  though  in  various  relative  amounts,  and  each 
proteose  contains  the  anti  as  well  as  the  hemi  group.  The  same  is  true  for 
the  peptone  obtained  in  pepsin  digestion,  hence  he  calls  it  ampho peptone. 
In  tryptic  digestion  a  cleavage  of  the  amphopeptone  takes  place  into  anti- 
peptone  and  hemipeptone.  Of  these  two  peptones  the  hemipeptone  is  further 
split  into  amino-acids  and  other  bodies,  while  the  antipeptone  is  not  attacked. 
By  the  sufficiently  energetic  action  of  trypsin  only  one  peptone  is  at  last 
obtained,  the  so-called  antipeptone. 

KtJHXE  and  his  pupils,  who  have  conducted  extensive  investigations 
on  the  proteoses  and  peptones,  classify  the  various  proteoses  according 
to  their  different  solubilities  and  precipitation  properties.  In  the  pepsin 
digestion  of  fibrin  *  they  obtained  the  following  proteoses:  (a)  Hetero- 
proteose,  insoluble  in  water  but  soluble  in  dilute  salt  solution;  (6)  Proto- 
proteose,  soluble  in  salt  solution  and  water.  These  two  proteoses  are 
precipitated  by  NaCl  in  neutral  solutions,  but  not  completely.  Hetero- 
proteose  may,  by  being  in  contact  with  water  for  a  long  time  or  by  drj-ing^ 
be  converted  into  a  modification,  called  (c)  Di/sproteose,  which  is  insoluble 


'  Pfliiger's  Archiv,  Si. 

^  See  Kiihne,  Verhandl.  d.  naturhistor  Vereins  zu  Heidelberg  (N.  F. ),  3;  J.  Wenz, 
Zeitschr.  f.  Biologie,  22;  Kiihne  and  Chittenden,  Zeitschr.  f.  Biologie,  22;  R.  Xeu- 
meister,  ibid.,  23;  Kiihne,  ibid.,  29. 

'  Schiitzenberger,  Bull,  de  la  Soc.  chimique  de  Paris,  23;  Kiihne,  Verhandl.  d. 
naturhist.  Vereins  zu  Heidelberg  (N.  F.),  1,  and  Kiihne  and  Chittenden,  Zeitschr,  f. 
Biologie,  19.     See  also  Paal,  Ber.  d.  deutsch.  chem.  Gesellsch.,  27. 

*  See  Kiihne  and  Chittenden,  Zeitschr.  f.  Biologie,  20. 


52  THE  PROTEIN  SUBSTANCES. 

in  dilute  salt  solutions,  (d)  Deuteroproteose  is  a  proteose  which  is  soluble 
in  water  and  dilute  salt  solution  and  which  is  incompletely  precipitated 
from  acid  solution  by  saturating  with  NaCl,  and  is  not  precipitated  from 
neutral  solutions.  This  precipitate  is  a  combination  of  the  proteose  with 
acid  (Herth^).  The  deuteroproteose  is  essentially  the  same  thing  that 
Brucke  has  designated  as  peptone. 

The  proteoses  obtained  from  different  protein  bodies  do  not  seem  to  be 
identical,  but  differ  in  their  behavior  to  precipitants.  Special  names  have 
been  given  to  these  various  proteoses  according  to  the  mother-protein, 
namely,  albumoses,  globuloses,  vitelloses,  caseoses,  myosinoses,  etc.  These 
various  proteoses  are  further  distinguished,  as  proto-,  hetero-,  and  deutero- 
caseoses,  for  example.  Chittenden  ^  has  suggested  the  common  name 
proteoses  for  the  products  formed  intermediary  between  the  proteins  and 
peptones  in  the  digestion  of  animal  and  vegetable  proteins.  We  have 
made  use  of  it  in  this  sense  in  preference  to  the  word  albumose  (which  is 
used  in  the  German  and  by  some  other  waiters),  but  which  will  be  used 
in  this  book  as  indicating  the  intermediary  products  in  the  hydrolysis 
of  albumins  and  not  as  a  general  term.  Certain  proteoses  have  also  been 
obtained  in  a  crystalline  state  (Schrotter). 

Neumeister  ^  designates  as  atmidalbumose  that  body  which  is  obtained  by 
the  action  of  superheated  steam  on  fibrin.  At  the  same  time  he  also  obtained  a 
substance  called  atmidalbumin,  which  stands  between  the  albuminates  and  the 
proteoses. 

Of  the  soluble  proteoses  Neumeister  designates  the  protoproteose  and 
heteroproteose  as  primary  proteoses,  while  the  deuteroproteoses,  which  are 
closely  allied  to  the  peptones,  he  calls  secondary  proteoses.  As  essential 
differences  between  the  primary  and  secondary  proteoses  he  suggests  the 
following:'*  The  primary  proteoses  are  precipitated  by  nitric  acid  in  salt- 
free  solutions,  while  the  secondary  proteoses  are  precipitated  only  in  salt 
solutions,  and  certain  deuteroproteoses,  such  as  deuterovitellose  and  deu- 
teromyosinose,  are  precipitated  by  nitric  acid  only  in  solutions  saturated 
with  NaCl.  The  primary  proteoses  are  precipitated  from  neutral  solutions 
by  copper-sulphate  solution  (2:100),  and  by  NaCl  in  substance,  while  the 
secondary  proteoses  are  not.     The  primary  proteoses  are  completely  pre- 

'  Monatshefte  f.  Chem.,  5. 

^  Kiihne  and  Chittenden,  Zeitschr.  f.  Biologie,  22  and  25;  Neumeister,  ibid.,  23; 
Chittenden  and  Hartwell,  Joum.  of  Physiol.,  11  and  12;  Chittenden  and  Painter, 
Studies  from  the  Lalx)ratory,  etc.,  Yale  University,  2,  New  Haven,  1887;  Chittenden. 
ibid.,  3;  Sebelien,  Chem.  Centralblatt,  1890;  Chittenden  and  Goodwin,  Joum.  of 
Physiol.   12. 

^  Zeitschr.  f.  Biologie,  26.  See  also  Chittenden  and  Meara,  Joum.  of  Physiol., 
15,  and  Salkowski,  Zeitschr.  f.  Biologie,  34  and  37. 

*  Neumeister,  Zeitschr.  f.  Biologie,  21  and  26. 


PROTEOSES  AND  PEPTONES.  53 

cipitated  from  a  solution  saturated  with  NaCl  by  the  addition  of  acetic 
acid  saturated  with  salt,  while  the  secondary  proteoses  are  only  partly 
precipitated.  The  primary  proteoses  are  readily  precipitated  by  acetic 
acid  and  potassium  ferrocyanide,  while  the  secondary  are  only  incompletely 
precipitated  after  some  time.  The  primary  proteoses  are  also,  according 
to  PiCK.i  completely  precipitated  by  ammonium  sulphate  (added  to  one- 
half  saturation),  while  the  secondary  proteoses  remain  in  solution. 

The  true  peptones,  as  they  were  formerly  considered  to  be,  are  exceed- 
ingly hygroscopic,  and  if  perfectly  dry,  sizzle  like  phosphoric  anhydride 
when  treated  with  a  little  water.  They  are  exceedingly  soluble  in  water 
diffuse  more  readily  than  the  proteoses,  and  are  not  precipitated  by  ammo- 
nium sulphate.  In  contradistinction  to  the  proteoses,  the  true  peptones 
are  not  precipitated  by  nitric  acid  (even  in  solutions  saturated  with  salt), 
by  sodium,  chloride  and  acetic  acid  saturated  with  salt,  potassium  ferro- 
cyanide and  acetic  acid,  picric  acid,  trichloracetic  acid,  potassium-mercuric 
iodide,  or  hydrochloric  acid.  They  are  precipitated  by  phosphotungstic  acid, 
phosphomolybdic  acid,  corrosive  sublimate  (in  the  absence  of  neutral  salts), 
absolute  alcohol,  and  tannic  acid,  but  the  precipitate  may  redissolve  on  the 
addition  of  an  excess  of  the  precipitant.  As  an  important  difference  between 
amphopeptone  and  antipeptone  we  must  also  mention  that  the  former  gives 
Millon's  reaction,  while  the  antipeptone  does  not. 

In  regard  to  the  precii^itation  by  alcohol  we  must  call  attention  to  the  observa- 
tions of  Frankel  that  not  only  are  the  acid  combinations  of  peptone  (Paal) 
soluble  in  alcohol,  but  also  the  free  peptone,  and  'Frankel  has  even  suggested  a 
method  of  preparation  based  on  this  behavior.  Schr5tter  -  has  also  prepared 
crystalline  proteoses  which  were  soluble  in  hot  alcohol,  especially  methyl  alcohol. 

The  views  on  the  hydrolytic  cleavage  products  of  peptic  and  tryptic 
digestion  which  were  accepted  until  a  few  years  ago  have  recently  been 
considerably  modified  in  several  points.  As  this  question  of  peptones 
is  at  the  present  time  undergoing  active  development,  and  as  it  is  also 
very  complicated  and  still  not  clear  in  many  points,  it  is  at  present  not 
possible  to  give  a  clear,  short,  and  still  comprehensive  discussion  of  the 
subject.     We  can  give  here  only  the  most  important  results. 

The  older  view  that  in  peptic  digestion  only  proteoses  and  peptones,  but 
no  simpler  cleavage  products,  are  formed  has  been  shown  not  to  be  true. 
The  works  of  Zunz,  Pfaundler,  Salaskin,  Law^row',  Langstein,^  and 
others  have  shown  that  simpler  products  can  be  produced,  some  whose 

■  Zeitschr.  f.  physiol.  Chem.,  2i. 

'  Frankel,  Zur  Kenntnis  der  Zerfallsprodukte  des  Eiweisses  bei  peptischer  und 
tryptischer  Verdauung,  Wien,  1896;    Schrotter,  Monatshefte  f.  Chem.,  14  and  10. 

'  Zunz,  Zeitschr.  f.  physiol.  Chem.,  28,  and  Hofmeister's  Beitrage,  2;  Pfaundler, 
Zeitschr.  f.  physiol.  Chem.,  30;  Salaskin,  ibid.,  32;  Salaskin  and  Kowalewsky,  ibid., 
38;  Lawrow,  ibid.,  33;  Langstein,  Hofmeister's  Beitrage,  1  and  2. 


54  THE  PROTEIN  SUBSTANCES. 

nature  is  still  unknown,  while  others  are  known,  such  as  alanine,  leucine, 
leucinimide,  aminovalerianic  acid,  aspartic  and  glutamic  acids,  phenyl- 
alanine, tyrosine,  pyrrolidine-carboxylic  acid,  and  lysine  and  on  further 
cleavage  indeed  also  oxyphenylethylamine,  tetra-  and  pentamethylene- 
diamine.  It  has  not  been  possible  to  cause  a  disappearance  of  the  biuret 
reaction,  and  the  occurrence  of  tryptophane  is  somewhat  disputed.  Mal- 
FATTi  obtained  tryptophane  in  peptic  digestion  only  when  he  used  a  certain 
apparently  impure  preparation  of  jiepsin,  and  on  using  pepsin  ])urified 
according  to  Pekelharing  it  was  absent.  According  to  Pekelharixg.^ 
purified  pepsin  also  yields  tryjjtophane  when  the  solution  is  rich  in  pepsin, 
and  also  when  the  acidit\'  is  not  too  strong,  in  the  presence  of  small  amounts 
of  pepsin. 

In  connection  with  the  above-mentioned  experimental  results  it  must  be 
remarked  that  not  all  the  products  found,  for  example  the  oxyphenylethylamine 
and  the  diamines,  are  produced  by  the  action  of  pepsin,  but  rather  by  the  action  of 
other  enzymes.  In  certain  cases,  undoubtedly,  impure  pepsin  was  used,  or  indeed 
autodigestion  of  the  stomach  was  carried  on,  and  the  action  of  other  enzymes 
was  not  excluded.  In  other  cases  the  digestion  with  pepsin  and  considerable 
acid  (even  1  per  cent  H2SO4)  was  continued  for  a  very  long  time,  indeed  for  an 
entire  year,  without  controlling  the  influence  of  the  acid  alone  upon  the  proteoses. 

KtJHNE's  view  that  in  tryptic  digestion  always  a  peptone,  so-called 
antipeptone,  remains  which  cannot  be  further  split  is  not  strictly  true. 
By  sufficiently  long  autodigestion  of  the  pancreas  Kutscher^  was  able 
to  obtain  as  final  products  a  mixture  of  digestion  products  which  failed  to 
respond  to  the  biuret  test.  In  this  connection  we  must  remark  that  the 
pure  antipeptone  (see  below),  isolated  by  Siegfried,  could  be  split  b}' 
trypsin  only  with  great  difficulty,  and  also  that  the  complete  disappearance 
of  the  biuret  reaction  in  tryptic  digestion  does  not  show  that  a  complete 
decomposition  into  amino-acids  has  taken  place.  According  to  E.  Fischer 
and  Abderhalden,^  polypeptide-like  bodies  are  produced,  especially  in 
tryptic  digestion,  and  these  bodies  resist  the  prolonged  action  of  the  enzyme^ 
but  yield  several  different  amino-acids  on  hydrolytic  cleavage  by  acids. 
The  same  is  probably  also  true  for  peptic  digestion  (see  below),  and  the 
difference  in  the  digestive  products  between  pepsin  and  trypsin  digestion 
consists  essentially  only  in  that  in  the  first  case  the  cleavage  is  slower  and 
does  not  proceed  so  far,  hence  the  i^iuret  reaction  remains  and  no  forma- 
tion of  tryptophane  takes  place. 

B}^  the  use  of  the  methods  specially  worked  out  by  the  Hofmeister 
s^chool,  of  fractionally  salting  out  with  ammonium  sulphate  or  zinc  sul- 

'  Malfatti,  Zeitschr.  f.  physiol.  Chem.,  31;  Pekelharing,  Archives  d.  scienc.  biolog. 
de  St.  P6tersbourg,  11;   Pawlow  Festband. 

"^  Zeitschr.  f.  physiol.  Chem.,  25,  26,  28,  and  Die  Endprodukte  der  Trypsin- 
verdauung,   Habilitationsschrift   Strassburg,   1899. 

^Zeitschr.  f.  physiol.  Chem.,  39. 


PROTEOSES  AND   PEPTONES.  55 

phate,  numerous  attempts  to  separate  the  various  proteoses  and  peptones 
have  recently  been  made  by  Umber,  Alexander,  Pfaundler,  and  espe- 
cially by  Pick  and  Zunz.^  Not  onh^  have  we  learned  by  these  methods  of  a 
larger  number  of  proteoses,  but  our  older  conception  of  the  products  formed 
primarily  has  been  materially  modified.  Immediately  at  the  commencement 
of  digestion,  even  in  ]:)eptic  digestion,  a  splitting  of  the  protein  molecule 
into  several  complexes  takes  place.  In  opposition  to  the  view  of  Huppert,^ 
that  the  proteoses,  in  pepsin  digestion,  are  always  derived  from  the  pri- 
marily formed  acid  albuminate.  Pick  and  Zuxz  have  shown  that  several 
proteoses,  as  well  as  acid  albuminate,  appear  as  primary  products  at  the 
commencement  of  the  digestion.  According  to  Goldschmidt  ^  a  splitting 
off  of  proteoses  and  the  formation  of  acid  albuminate  takes  place  simul- 
taneously b}^  the  action  of  dilute  acids  alone.  Besides  the  proteoses  we 
have,  according  to  Zuxz  and  Pfauxdler,  even  at  the  beginning,  also 
other  primary  bodies,  which  cannot  be  salted  out  and  which  do  not  give 
the  biuret  reaction,  but  are  in  part  precipitated  by  phosphotungstic  acid. 
These  little  known  products  seem  to  be  intermediate  between  the  pep- 
tones and  the  amino-acids,  and  they  correspond  probably  to  the  poly- 
peptide bodies  obtained  by  Fischer  and  Abderhaldex  in  tryptic  digestion. 

By  fractional  precipitation  of  Witte's  peptone  with  ammonium  sulphate 
Pick  has  obtained  various  chief  fractions  of  proteoses.  The  first  contains  the 
proto-  and  heteroproteoses,  whose  precipitation  limit  lies  at  24-42  per  cent  satu- 
ration with  ammonium  sulphate  solution,  i.e.,  the  presence  of  2-4-42  c.c.  of  the 
saturated  ammonium  sulphate  solution  in  100  c.c.  of  the  liquid.  Then  follows 
a  fraction  A  at  54-62  per  cent  saturation,  then  a  third  fraction  B,  with  70-9.5 
per  cent  saturation,  and  finally  fraction  C,  which  precipitates  from  the  saturated 
solution  on  acidification  with  sulphuric  acid  saturated  with  the  salt. 

The  hetero-  and  protoproteoses  are  not,  according  to  our  present  views, 
the  only  primary  proteoses.  In  the  proteose  fraction  B,  obtained  on  saturat- 
ing with  ammonium  sulphate  in  neutral  liquids,  primary  proteoses  are 
also  found.  As  examples  we  may  mention  the  glucoproteose  (Pick)  which 
contains  a  carbohydrate  group,  and  Hofmeister's  ■*  synproteose.  Unequal 
responsiveness  to  the  salting-out  process  is  no  longer  sufficient  to  differen- 
tiate between  the  primary  and  secondary-  proteoses,  especially  as  Haslam  ^ 
has  shown  that  the  products  obtained  by  fractionation  are  not  units  l)ut 

'  Umber,  Zeitschr.  f.  phj'siol.  Chem.,  2o;  Alexander,  ibuL,  25;  Pfaiindler,  ibid., 
30;  Zunz,  ibid.  28,  and  Hofmeister's  Beitrage,  2;  Pick,  ibid.,  2,  and  Zeitschr.  f.  physiol. 
Chem.,  24  and  28. 

^  Schiitz  and  Huppert.  Pfliiger's  Arch.,  80. 

'  F.  Goldschmidt,  Ueber  die  Einwirkiing  von  Sauren  auf  Eiweissstoffe,  Inaug,- 
Diss.  Strassburg,  1898. 

^  Ueber  Bau  imd  Gnippirung  der  Eiweisskorper,  Ergebnisse  der  Physiol.,  Jahrg.  I 
Abt.  1,  78.3.  "  ' 

*  Journ.  of  Physiol.,  32. 


56  THE  PROTEIN  SUBSTANCES. 

mixtures.     According  to  Haslam,  it  is  possible  to  separate  the  various  pro- 
teoses by  salting  out  only  by  following  the  directions  he  suggests. 

Under  these  circumstances  we  cannot  enter  into  a  discussion  of  the 
properties  of  various  proteoses  thus  far  prepared.  The  differences  which 
exist  between  the  hetero-  and  protoproteoses  obtained  from  fibrin  (Pick^) 
are  of  great  interest.  The  heteroproteose  is  insoluble  in  32  per  cent  alcohol^ 
yields  only  very  little  tyrosine  or  indol,  but  gives  abundant  leucine  and 
glycocoU,  and  contains  about  39  per  cent  of  the  total  nitrogen  in  a  basic 
form.  The  protoproteose,  on  the  contrary,  is  soluble  in  80  per  cent  alcohol, 
yields  considerable  tyrosine  and  indol,  only  little  leucine  but  no  glycocoU,. 
and  contains  only  about  25  per  cent  basic  nitrogen.  Friedmanx,  Hart^ 
and  Levene  have  obtained  very  similar  results  in  regard  to  the  quantity 
of  basic  nitrogen  in  the  two  proteoses,  although  Levene  did  not  find  the 
same  results  as  Pick  in  regard  to  the  amounts  of  leucine,  tyrosine,  and 
glycocoll  in  the  two  proteoses.  However,  Haslam  has  shown  that  Pick's 
heteroproteose  was  contaminated  by  a  proteose  which  was  readily  pre- 
cipitated by  alcohol  and  called  a-protoproteose  and  that  there  also  exists 
Ijesides  this  a  second  protoproteose,  /?-protoprot€ose,  which  is  probably 
identical  with  Pick's  proteose;  still  this  does  not  change  the  fundamental 
fact  that  we  have  a  hetero-  and  a  protoproteose  which  differ  essentially 
in  chemical  constitution.  Hart^  also  showed  that  the  heteroproteose 
(from  muscle  syntonin)  is  considerably  richer  in  arginine  and  poorer  in 
histidine  than  the  protoproteose. 

According  to  Pick,  the  heteroproteose  is  also  more  resistant  towards 
trypsin  digestion  than  the  protoproteose,  a  behavior  which  coincides  with 
KtJHNE's  view  of  a  resistant  atomic  complex,  an  antigroup,  in  the  protein 
bodies.  Kuhne  and  Chittenden  ^  obtained  regularly  on  the  trjqDtic 
digestion  of  heteroproteose  a  separation  of  so-called  antialbumid,  a  body 
which  is  attacked  with  great  difficulty  in  tryptic  digestion,  but  which  sepa- 
rates as  a  jelly-like  mass  and  which  is  richer  in  carlDon  (57.5-58.09  per  cent), 
but  poorer  in  nitrogen  (12.61-13.94  per  cent),  than  the  original  protein. 

This  antialbumid  has  recently  attracted  further  attention,  because,  as 
first  found  by  Danilewsky  and  as  other  investigators,  Okunew,  Saw- 
JALOW,  Lawrow,  and  Salaskin  and  Kurajeff,  have  further  shown, 
solutions  of  rennin,  gastric  juice,  pancreatic  juice,  and  papain  cause  a 
coagulum  in  not  too  dilute  proteose  solutions.  These  coagula,  called 
plasteines  (coagulum  1)}^  rennin)  by  Sawjat.ow,  and  coaguloses  (coagu- 
lum by  papain)  by  Kurajeff,^  are  similar  in  many  respects  to  antialbumid, 

'  Zeitschr.  f.  pliysiol.  Chem.,  28. 

^  P'riedmann,  ibul.,  21);  Hart,  ibid.,  33;  Haslam,  1.  c;  Levene,  Joum.  of  BioL 
Chem.,  1. 

^  Kiihne  and  Chittenden,  Zeitschr.  f.  Biologie,  19,  20. 

*  The  works  of  Danilewsky  and  Okunew  are  cited  and  reviewed  in  the  following: 


PLASTEINES.  57 

having  a  higher  content  of  carbon  (57-60  per  cent)  and  nitrogen  (13-14.6 
per  cent).  They  are  produced  only  from  proteoses  and  not  from  peptones, 
and  form  only  a  small  fraction  of  the  related  proteose.  We  cannot  state 
anything  with  positiveness  at  present  in  regard  to  their  importance.  It 
is  evident  from  their  composition  that  they  do  not  represent  the  reforma- 
tion of  protein  from  the  proteoses,  as  claimed  by  some  investigators,  and 
their  protein  natm'e  has  indeed  been  disputed. 

By  fractional  treatment  of  Witte's  peptone  with  alcohol  and  acetone 
H.  Bayer  ^  has  shown  that  the  substance  plasteinogen,  from  which  plastein 
is  produced,  is  not  a  true  protein.  This  substance  was  soluble  in  alcohol- 
acetone  and  gave  on  further  purification  neither  the  Millon  reaction  nor 
the  biuret  test.  Its  composition  also  differs  from  the  proteins,  contain- 
ing C  38.43,  H  7.01,  N  8.05  per  cent,  and  the  relation  of  C:N  was  4.755:1. 
According  to  these  investigations  plasteinogen  is  not  a  proteose  but  may 
rather  be  considered  as  a  peptoid. 

It  is  also  generally  admitted  that  the  peptones  are  mostly  mixtures 
of  various  bodies.^  Only  those  peptones  isolated  by  Siegfried  and  his 
pupils  MiJHLE,  Fr.  JMiJLLER,  BoRKEL,  Krijger,  and  ScHEERMEssER  ^  must 
be  considered  as  chemical  individuals.  All  these  peptones  have  a  pro- 
nounced acid  character  and  form  salts  with  carbonates  with  the  evolution 
of  carbon  dioxide;  they  are  levorotatory  and  show  a  constant  degree  of 
rotation.  The  pepsin-fibrin  peptones,  a  and  /?,  isolated  and  studied  by 
Siegfried,  ^Iijhle,  and  Borkel,  have  the  formulae  C21H34N6O9  and 
CoiHseNfiOio,  respectively.  The  /J-peptone  seems  to  be  converted  into 
a-peptone  on  the  splitting  off  of  water.  These  pepsin  peptones  give  the 
biuret  test  as  well  as  ]Millon's  reaction.  Their  solutions  are  not  pre- 
cipitated by  tannic  acid,  picric  acid,  corrosive  sublimate,  phosphotungstic 
acid,  or  alcohol,  but  are  precipitated  by  basic  lead  acetate,  metaphosphoric 
acid,  and  acetic  acid  and  potassium  ferrocyanide.  The  pepsin  peptone 
may  be  considered  as  an  amphopeptone  in  Kuhne's  sense,  for  in  trypsin 
digestion  amino-acids  are  formed,  and  all  the  tyrosine  and  arginine  are 
split  off  and  antipeptone  is  formed.  The  a-pepsin-fibrin  peptone  is,  like 
the  pepsin-glutin  peptone,  a  tribasic  acid  as  well  as  a  biacidic  base 
(Neumann)  .4 

The  trypsin-fibrin  antipeptones  studied  by  Siegfried  and  Muller  have 

Sawjalow,  Pfliiger's  Arch.,  85,  and  Centralbl.  f.  Physiol.,  Ifi;  Lavvrow  and  Salaskin, 
Zeitschr.  f.  physiol.  Chem.,  36;  Kurajeff,  Hofmeister's  Beitrage,  1  and  2;  see  also 
Sacharow,  Biochem.  Centralbl.,  1,  233. 

'  Hofmeister's  Beitrage,  4. 

^  See  Kutscher,  1.  c;  Friiiikel  and  Langstein,  Wien.  Sitzungsber.  Math.-Naturw. 
Klasse,  110,  1901;  Pick,  Hofmeister's  Beitrage,  2. 

^Siegfried,  Arch.  f.  (Aaat.  u.)  Physiol.,  1894;'  also  Zeitschr.  f.  physiol.  Chem.,  21, 
43,  and  4n;  Siegfried  and  his  pupils,  ibid.,  38;   Scheermesser,  ibid..  41. 

*  Zeitschr.  f.  physiol.  Chem.,  45. 


58  THE  PROTEIN  SUBSTANCES. 

the  formulae  a,  C10H17N3O5,  and  /?,  C11H19N3O5.  They  have  a  different 
specific  rotation  and  are  at  the  same  time,  according  to  Neumann,  bibasic 
acids  and  monoacidic  bases.  The  fact  that  two  different  antipeptones  are 
formed  from  the  pepsin-fibrin  peptone  shows  that  this  latter  contains  at 
least  two  antigroups,  and  not,  as  Kijhne  claimed,  only  one.  The  antipep- 
tones do  not  give  the  biuret  test,  but  respond  to  Millon's  reaction,  and 
contain  no  tyrosine  groups.  They  are  precipitated  by  alcohol,  but  are  pre- 
cipitated less  readily  or  less  completely  by  the  reagents  which  precipitate 
the  pepsin  peptones.  They  have  a  persistent  resistance  towards  further 
cleavage  by  trypsin.  On  hydrolysis  with  mineral  acids  they  yield  arginine, 
lysine,  glutamic  acid,  and  it  seems  also  aspartic  acid.  The  quantity  of 
basic  nitrogen  is  less  than  2.5  per  cent,  and  the  nitrogen  split  off  as  ammonia 
in  antipeptone  is  /?  16.1  and  a  21.9  per  cent  of  the  total  nitrogen. 

The  glutin  peptones  isolated  by  Siegfried  and  Kruger  have  the  formulae 
C21H39N7O10  for  the  pepsin-glutin  peptone  and  C19H30N6O9  for  the  /?-tryp- 
gin-glutin  peptone.  The  composition  of  the  apparently  very  pure  pepsin- 
glutin  peptone  as  prepared  by  Scheermesser  was  C21H39N7O10.  It  gave 
the  biuret  test,  but  did  not  give  the  other  protein  color  reactions.  Its 
solution  became  cloudy  with  picric  acid  but  was  not  precipitated.  Tannic 
acid  gave  a  precipitate  which  was  soluble  in  acetic  acid,  while  phosphotung- 
stic  acid  produced  a  precipitate  only  in  concentrated  solution.  Of  the  total 
nitrogen  25  per  cent  existed  as  basic  and  69.85  per  cent  as  amino-acid 
nitrogen.  It  yielded  arginine,  lysine,  glutamic  acid,  and  glycocoU  as  hydro- 
xy tic  cleavage  products;  this  peptone  contained  no  histidine. 

From  glutin  peptone,  Siegfried,  on  warming  with  hydrochloric  acid, 
obtained  a  base,  C21H39N9O8,  which  can  also  be  directly  obtained  from 
gelatine.  This  he  calls  a  kyrin  because  it  is  to  be  considered  as  a  basic 
protein  nucleus,  and  he  calls  this  special  one  glutokyrin.  The  glutokyrin 
gives  the  biuret  reaction  and  is  considered  as  a  basic  peptone.  On  com- 
plete hydrolytic  cleavage  it  yields  arginine,  lysine,  glutamic  acid,  and 
glycocoll.  Of  the  total  nitrogen  two-thirds  belongs  to  the  bases  and  one- 
third  to  the  amino-acids.  Similar  basic  nuclei,  protokyrins,  have  recently 
been  obtained  by  Siegfried  1  from  fibrin  and  casein,  using  the  same  method. 
Casein okyrin  gives  a  non-crystalline  sulphate  but  a  crystalline  phospho- 
tungstate.  The  free  caseinokyrin  has  an  alkaline  reaction,  gives  the  biuret 
test,  and  its  composition  corresponds  to  the  formula  C23H47N9O8.  It 
yields  arginine,  lysine,  and  glutamic  acid  on  cleavage.  The  basic  nitro.aen 
amounts  to  about  85  per  cent  of  the  total  nitrogen,  and  hence  caseinokyrin 
behaves  in  this  respect  like  a  protamine. 

Skraup  and  Zwerger  have  presented  certain  doubts,  based  upon  their 

*  Kgl.  Sachs.  Ges.  d.  Wiss.,  Math.-Phys.  Klasse,  1903,  and  Zeitschr.  f.  physiol. 
Chem.,   43. 


BASIC  NUCLEUS  OF  THE   PROTEINS.  59 

own  investigations,  as  to  the  individuality  of  the  products  designated 
kvrins  by  Siegfried.  Siegfried  ^  repudiates  the  claims  made  by  thase 
investigators,  criticises  their  methods  and  presents  new  claims  for  the 
indi\aduality  of  the  kyrins,  describing  exactly  the  properties  of  their  phos- 
photungstates  and  picrates.  The  constant  composition  of  the  sulphate 
is  of  importance.  It  was  obtained  thus  only  after  repeated  recr\-stalliza- 
tion  (reprecipitation  nine  times  of  a  caseinokyrin  sulphate),  but  it  remained 
unchanged  on  recrystallization  up  to  fifteen  times. 

Among  the  known  clea\'age  products  of  proteins,  arginine  is  the  only 
one  which,  up  to  the  present,  is  never  absent,  and  for  this  reason  we  desig- 
nate as  proteins  only  those  atomic  complexes  wliich  contain,  tesides  chained 
monamino-acids,  also  arginine,  or,  more  simply,  show  the  above  two  kinds 
of  imide  bindings.  Hence  caseinokyrin.  which  yields  only  arginine,  lysine, 
and  glutamic  acid,  and  scombrin  (see  below),  which  yields  only  arginine, 
a-pyrrolidine-carboxyUc  acid,  and  alanine  are  the  simplest  known  proteins. 

Scombrin  belongs  to  the  group  of  substances  called  protamines,  which 
will  be  treated  of  later,  and  these  substances  are  strongly  basic,  simple  of 
constitution,  give  the  biuret  reaction,  and  are  similar  to  the  proteids. 
According  to  Kossel^  we  can  conceive  of  the  formation  of  the  protamines 
by  a  successive  cleavage  of  the  typical  proteins,  and  the  occurrence  of 
basic  protokyrins  in  the  hydrolytic  cleavage  of  genuine  proteins  like  gelatine 
has  given  valuable  support  to  Kossel's  theor}'  as  to  a  basic  nucleus  in 
the  protein  bodies.  We  must  not  infer  from  tliis  that  each  protein  con- 
tains only  one  nucleus.  It  is,  on  the  contrary',  possible  and  not  improbable 
that  each  protein  is  composed  of  several  larger  complexes  and  that  each 
of  these  contains  a  special  nucleus.  The  proteoses  may  be  considered  as 
large  complexes  of  this  kind  which,  at  least  in  part,  do  not  separate  but  seem 
to  stand  together.  The  cleavage  of  proteins,  according  to  ScHiJrzENBERGER 
and  Kt'HXE,  into  a  hemi-  and  an  antigroup,  of  which  the  first  contains, 
among  other  complexes,  the  readily  split  tyrosine  and  tiyptophane,  while 
the  antigroup  contains  a-proUne  (a-pyrrolidine-carboxyUc  acid),  glycocoU, 
and  phenylalanine;  the  different  beha\'iors  of  proto-  and  heteroproteoses ; 
and  the  occurrence  of  non-biuret-gi\-ing  polypeptides  in  digestion,  coincide 
well  ^^ith  such  a  view. 

On  account  of  the  cleavage  taking  place  in  digestion,  the  digestive 
products  should  have  a  lower  molecular  weight  than  the  original  protein. 
This  is  really  the  case.  The  molecular  weight  of  the  different  proteins  has 
not  been  determined  with  certainty .^  but  it  is  generally  considered  as  about 

•  Skraup  and  Zwerger,  Monatshefte  f.  Cheniie,  26;    Siegfried,  Zeitschr.  f.  Physiol 
48. 

-  Zeitschr.  f.  physiol.  Chem.,  44. 

'See  especially  F.  N.  Schulz,  Die  Grosse  des  EiweissmolekiiLs,  Jena,  1903. 


60  THE  PROTEIN  SUBSTANCES. 

5000-10  000  for  the  albumins  and  for  casein.  The  molecular  weight  for 
protoproteoses  was  found  by  Sabanejew  to  be  2467-2643,  and  3200  for 
the  deuteroproteoses.  The  peptones  have  a  still  lower  molecular  weight, 
being  between  400  and  250  for  the  different  peptones  (Sabanejew,  Paal, 
Sjoqvist  1). 

The  elementary  analj^ses  ^  have  not  given  us  much  information  as  to 
the  characteristic  differences  between  the  various  proteoses  and  most 
so-called  peptones.  Certain  proteoses,  especially  those  that  can  be  salted 
out  with  difficulty,  and  the  peptones  differ  very  materially  in  composition 
from  the  mother-substances  and  often  have  a  lower  carbon  content. 

Besides  the  behavior  in  the  salting-out  process,  attempts  have  been  made  to 
find  other  points  of  difference  between  the  peptones  and  proteoses.  Schrotter 
and  Frankel  ^  consider  the  sulphur  content  as  a  pronounced  point  of  difference. 
The  peptones,  according  to  them,  are  free  from  sulphur,  while  the  proteoses,  on 
the  contrary,  contain  sulphur.  Frankel  has  been  able  to  find  only  one  proteose 
(in  KiJHNE's  sense)  which  did  not  contain  sulphur. 

In  the  preparation  and  separation  of  various  proteoses  and  peptones  all 
precipitable  protein  is  always  removed  first  by  neutralization  and  then  by 
boiling.  The  proteoses  may  then  be  separated  from  the  peptones  by  means 
of  ammonium  sulphate  according  to  KtJHNE's  method,  and  divided  into 
different  fractions  according  to  the  method  of  Pick  and  the  Hofmeister 
school.  The  separation  and  preparation  of  pure  hetero-  and  protopro- 
teoses can  be  best  performed  by  the  method  suggested  by  Pick,  but  this 
method,  as  well  as  that  with  ammonium  sulphate,  gives  good  results  only 
when  the  precautions  suggested  by  Haslam*  are  carefully  followed.  As 
most  of  the  older  methods  do  not  give  pure  substances  but  rather  mixtures, 
it  is  perhaps  sufficient  simply  to  call  attention  here  to  other  methods, 
such  as  those  suggested  by  K.  Baumann  and  Bomer,  P.  ^Muller,  Frankel, 
Schrotter,  and  Paal.  The  only  method  which  seems  thus  far  to  have 
led  to  a  pure  preparation  of  peptone  is  the  iron  method  used  by  Siegfried.^ 

For  the  detection  of  proteoses  and  peptones  in  animal  fluids  we  proceed 
as  follows,  according  to  Devoto:  The  coagulable  proteins  are  removed  by 
prolonged  heating,  and  the  solution  is  then  saturated  with  ammonium  sul- 
phate. True  peptones  (besides  deuteroproteose  not  precipitated)  may  be 
detected  in  the  cold  filtrate  by  means  of  the  biuret  test.     The  proteoses 


'  Sabanejew,  Ber.  d.  d.  chem.  Gesellsch.,  20,  385;  Paal,  ibid.,  27,  1827;  Sjoqvist, 
Skand.  Arch.  f.  Physiol.,  5. 

^  Elementary  analyses  of  proteoses  and  peptones  will  be  found  in  the  works  of 
Kiihne  and  Chittenden  and  their  pupils,  cited  in  foot-note  2,  p.  52;  also  by  Herth, 
Zeitschr.  f.  physiol.  Chem.,  1,  and  Monatshefte  f.  Chem.,  5;  Maly,  Pfliiger's  Arch., 
9,  20;    Hcnninger,  Compt.  rend.,  80;   Schrotter,  1.  c;    Paal,  1.  e. 

^  Schrotter,  Monatshefte  f.  Chem.,  14  and  10;  Frankel,  Zur  Kenntnis  der  Zerfalls- 
produkte  des  Eiweiss  bei  peptischer  und  tryptischer  Verdauung,  Wien,  1896. 

*  Kiihne,  Zeitschr.  f.  Biologic.  28;   Pick,  I.  c;  Haslam,  1.  c. 

^  Baumann  and  Bomer,  Chem.  Centralbl.,  1898,  1,  640;  Miiller,  Zeitschr.  f.  physiol. 
Chem.,  20;  Frankel,  1.  c.,  Zur  Kenntnis,  etc.;  Schrotter,  Monatshefte  f.  Chem.,  14 
and  10;  Paal,  1.  c;  Siegfried,  1.  c. 


HISTONES.  61 

are  contained  in  the  mixture  of  precipitate  and  salt  crystals  collected  on 
the  filter.  The  proteoses  are  dissolved  from  this  mixture  by  washing  with 
water,  and  may  be  detected  in  the  wash-water  by  means  of  the  biuret 
test.  According  to  Halliburton  and  Colls  ^  traces  of  proteoses  may 
be  formed  from  other  proteins  in  this  method  by  prolonged  heating.  As 
the  best  method  they  suggest  either  the  precipitation  of  the  native  proteids 
b}'  the  addition  of  a  10  per  cent  trichloracetic-acid  solution,  or  the  con- 
version of  the  native  proteins  to  the  insoluble  form  by  the  continued  action 
of  alcohol.  The  last  method  is  hardly  applicable  to  blood-serum,  as  the 
so-called  fibrin-ferment,  which  also  gives  the  biuret  test,  is  not  made  insol- 
uble by  this  procedure. 

If  a  solution  saturated  with  ammonium  sulphate  is  to  be  tested  for  the 
biuret  reaction,  it  must  first  be  treated  with  a  slight  excess  of  concentrated 
caustic-soda  solution,  the  solution  being  kept  cold,  and  after  the  sodium 
sulphate  has  settled,  the  liquid  is  treated  with  a  2  per  cent  solution  of  copper 
sulphate,  drop  by  droj). 

The  estimation  of  nitrogen,  the  biuret  test  rcolorimetric),  and  the  polari- 
scopic  method  have  been  used  in  the  quantitative  estimation  of  proteoses 
and  peptones.    These  two  last-mentioned  methods  do  not  yield  exact  results. 

Coagulated  Proteins.  Proteins  ma}^  be  converted  into  the  coagulated 
condition  by  different  means:  by  heating,  b}"  the  action  of  alcohol,  especially 
in  the  presence  of  neutral  salts,  by  chloroform,  ether,  and  metallic  salts, 
and  by  the  prolonged  shaking  of  their  solutions  (Ramsdex^)^  and  in  cer- 
tain cases,  as  in  the  conversion  of  fibrinogen  into  fibrin  (Chapter  VI),  by  the 
action  of  an  enzyme.  The  nature  of  the  processes  which  take  place  during 
coagulation  is  unknown.  The  coagulated  albuminous  bodies  are  insoluble 
in  water,  in  neutral  salt  solutions,  and  in  dilute  acids  or  alkalies,  at  normal 
temperature.  They  are  dissolved  and  converted  into  albuminates  by 
the  action  of  less  dilute  acids  or  alkalies,  especially  on  heating. 

Coagulated  proteins  also  seem  to  occur  in  animal  tissues.  We  find,  at 
least  in  many  organs  such  as  the  liver  and  other  glands,  proteins  which  are 
not  soluble  in  water,  dilute  salt  solutions,  or  verv'  dilute  alkalies,  and  only 
dissolve  after  being  modified  by  strong  alkalies. 

Histones  are  basic  proteins  which  stand  to  a  certain  extent  between 
the  strongly  l^asic  protamines  (see  below)  and  the  tme  proteins.  Their 
content  of  nitrogen  varies  between  16.5  and  19.8  per  cent  and  in  certain 
instances  is  not  higher  than  in  other  proteins,  especially  vegetable  proteins. 
According  to  Kossel  and  Kutscher  and  Lawrow  they  are,  on  the  contrary^ 
richer  in  basic  nitrogen  and  especially  yield  more  arginine  than  other  proteins. 
Kossel  first  isolated  a  peculiar  protein  substance  from  the  red  corpuscles 
of  goose  blood  which  was  precipitated  by  ammonia,  and  because  of  its 
similarity  in  certain  regards  to  the  peptones  (in  the  old  sense)  he  called 

*  Devoto,  Zeitschr.  f.  physiol.  Chem.,  15;  Halliburton  and  Colls,  Joum.  of  Path, 
and  Bact.,  1895. 

2  Arch.  f.  (Anat.  u.)  Physiol.,  1894. 


62  THE  PROTEIN  SUBSTANCES. 

it  histone.  At  the  present  time  a  number  of  very  different  bodies  are 
described  as  histones,  such  as  those  obtained  from  nucleohistone  (Ltliex- 
feld),  from  hannoglobin  (globin  according  to  Schulz).  from  mackerel  si)er- 
matozoa  (scombron  according  to  Bang),  from  the  codfish  (gadushistor.e 
according  to  Kossel  and  Kutscher),  from  the  burbot,  (lotahistone.  Ehr- 
STROm),  and  from  the  sea-urchin  (arbacin,  jMathews).^ 

Sulphur  has  been  found  in  those  histones  in  which  it  has  been  tested  for. 
They  give  the  biuret  test,  but  as  a  rule  only  a  faint  ]\Iillon's  reaction.  The 
goose-blood  histone  first  studied  by  Kossel  gives  the  three  following  reac- 
tions: The  neutral  salt-free  solution  first,  does  not  coagulate  on  boiling; 
second,  gives  a  precipitate  with  ammonia  which  is  insoluble  in  an  excess 
of  the  precipitant;  third,  gives  a  precipitate  with  nitric  acid  which  disap- 
pears on  heating  and  reappears  on  cooling. 

The  different  histones  behave  differently  in  these  three  reactions, 
and  hence  they  are  not  specific.  On  the  other  hand,  all  histones  seem  to 
be  precipitated  from  neutral  solution  by  alkaloid  reagents,  and  they  also 
produce  precipitates  in  protein  solutions.  These  two  reactions  are  likewise 
not  specific  for  the  histones,  as  the  protamines  have  a  similar  behavior.  The 
histones  differ  from  the  protamines  by  having  a  much  lower  content  of  basic 
nitrogen,  and  also  probably  by  always  containing  sulphur.  Time  proteins, 
as  Osborne's 2  edestan,  also  give  these  two" reactions;  therefore  it  is  im- 
possible by  qualitative  tests  alone  to  identify  a  substance  as  a  histone 
with  posit iveness.  The  large  content  of  basic  nitrogen  and  of  argininc  is 
not  a  sure  point  of  difference  between  histones  and  other  bodies.  Histone 
yields  little  more  than  40  per  cent  basic  nitrogen,  while  a  heteroproteose 
yields  about  the  same,  namely,  39  per  cent.  Histone  yields  14-15.5  p:r 
cent  arginine  (gadushistone),  and  the  lotahistone  only  12  per  cent.  The 
plant -globulin  edestin  ^  yields  a  much  larger  amount  of  arginine,  namely, 
14.0?  per  cent.  On  hydrolytic  cleavage  the  histones,  like  other  proteins, 
but  unlike  the  protamines,  yield  a  large  number  of  monamino-acids. 
Abderhalden  and  Rona^  obtained  from  thymus  histone  the  following: 
leucine  11.8,  alanine  3.46,  glycocoll  0.50,  a-proline  1.46,  phenylalanine  2.20, 
tvrosine  5.20,  and  glutamic  acid  0.53  per  cent.  According  to  Kossel  the  his- 
tones are  probably  intermediate  bodies  between  the  protamines  and  pro- 
tein bodies  on  the  demolition  of  the  latter,  and  if  this  is  true,  then  it  is  not 


1  Kossel,  Zf'itschr.  f.  physiol.  Chem.,  8,  and  Sitzungsber.  der  Gosellsch.  zur  Beford. 
d.  ges.  Naturwiss.  zu  Marburg,  1S97;  Kossel  and  Kutscher,  ibid.,  1900,  and  Zeitschr. 
f.  physiol.  Chem.,  31;  Lawrow,  i6m/.,  28,  and  Ber.  d.  d.  chem.  Gesellsch.,  34;  Lilienfeld, 
Zeitschr.  f.  physiol.  Chem.,  18;  Schulz,  ibid.,  24;  Bang,  ibid.,  27;  Ehrstrom,  ibid., 
32;    Mathews,  ibid.,  28. 

2  Zeitschr.  f.  physiol.  Chem.,  33. 

3  See  Kossel,  Ber.  d.  d.  chem.  Gesellsch.,  34,  3236. 
"  Zeitschr.  f.  physiol.  Chem.,  41, 


PROTA^IINES.  63 

to  be  expected  that  histones  should  have  perfectly  specific  reactions,  and 
for  this  reason  it  is  hardly  possible  for  the  present  to  give  a  precise  defi- 
nition for  the  histones. 

The  parahistone  found  by  Fleroff  in  the  thymus  gland  yields  so  little  basic 
nitrogen  that  it  probably  does  not  belong  to  the  histone    group   (Kossel  and 

KUTSCHER  ^). 

Protamines.  In  close  relationship  to  the  proteins  stands  a  group  of 
substances,  the  protamines,  discovered  by  Miescher,  which  are  desig- 
nated by  Kossel  as  the  simplest  proteins  or  as  the  nucleus  of  the  jDro- 
tein  bodies.  Thus  far  they  have  been  found  only  in  comljination  with 
nucleic  acids  in  fish  spermatozoa.  They  differ  essentially  from  the  proteins 
by  the  fact  that  they  yield  chiefly  diamino-acids  (always  abundant  arginine) 
as  cleavage  products,  and  oidy  a  small  amount  of  monamino-acids.  They 
are  strongly  basic  substances  rich  in  nitrogen  (about  30  per  cent  or  more) 
and  have  high  molecular  weight. 

Protamine  was  discovered  by  Miescher  2  in  salmon  spermatozoa.  Later 
Kossel  isolated  and  studied  similar  bases  from  the  spermatozoa  of  herring, 
sturgeon,  mackerel,  and  other  fishes.  As  all  these  bases  are  not  identical, 
Kossel  uses  the  name  protamines  to  designate  the  group  and  calls  the 
individual  protamines  according  to  their  origin  salmine,  clupeine,  scomhritie, 
sturine,  cyprinine,  cyclopterine,  etc. 

The  percentage  composition  of  these  bodies  has  not  been  satisfactorily 
determined.  As  probable  formulae  we  have  for  salmine  C32H56N18O4 
(Miescher-Schmiedeberg)  or  C30H57N17O6  (Kossel  and  Goto),  for  clu- 
peine C30H62N14O9,  and  for  sturine  C36H69N19O7  (Kossel)  or  C34H71N17O9 
(Goto).  On  boiling  with  dilute  mineral  acids,  as  also  by  tryptic  digestion, 
the  protamines  first  yield  peptone-like  substances  called  protones,  from 
which  simpler  products  are  derived  on  further  cleavage.  All  protamines 
yield  arginine,  the  four  protamines  salmine,  clupeine,  cyclopterine,  and 
sturine  yielding  87.4,  82.2,  62.5,  and  58.2  per  cent  respectively.  Sturine 
yields  besides  this  the  two  hexone  bases  lysine,  12  per  cent,  and  histidine, 
12.9  per  cent.  Histidine  has  not  been  found  in  any  other  protamine.  The 
carp  protamine,  cyprinine,  occurs  in  two  different  modifications,  namel}', 
a-  and  ^9-cyprinine.     The  a-cyprinine  yields  only  little  arginine,  4.9  per 

'  Fleroff,  Zeitschr.  f.  physiol.  Chem.,  2S;    Kossel  and  Kutscher,  1.  c. 

^  In  regard  to  protamines,  see  Miescher,  Histochemische  und  Physiologische  Ar- 
beiten,  Leipzig,  1897;  Piccard,  Ber.  d.  deutsch.  chem.  Gesellsch.,  7;  Schmiedeberg, 
Arch.  f.  exp.  Path.  u.  Pharm.,  3";  Kossel,  Zeitschr.  f.  physiol.  Chem.,  22  (Ueber  die 
basischen  Stoffe  des  Zellkerns),  25,  165  and  190,  26,  40,  and  44,  and  Sitzungsber.  der 
Gesellsch.  zur  Beford.  der  ges.  Naturwiss.  zu  Marburg,  1897;  Berl.  klin.  Wochenschr., 
1904;  Kossel  and  Mathews,  Zeitschr.  f.  physiol.  Chem.,  23  and  25;  Kossel  and  Kutscher, 
ibid.,  SI;  (^-nto,  ibid.,  S7 ;  Kurajeff,  i6id.,  32;  Morkowin,  *«/.,  28;  Kossel  and  Dakin, 
iow/.,  40,  41,  and  44. 


64 


THE  PROTEIN  SUBSTANCES. 


€ent,  but  the  lysine  content  is  pronounced,  28.8  per  cent.  Of  the  total 
nitrogen  30.3  per  cent  exists  as  lysine.  Kossel  and  Dakin  have  obtained 
from  salmine  the  following  cleavage  products,  namely,  arginine  87.4, 
serine  7.8,  aminovalerianic  acid  4.3,  and  a-pyrrolidine-carboxylic  acid 
11  per  cent,  and  according  to  them  the  salmine  contains  about  10  mol.  ■ 
arginine,  2  mol.  serine,  1  mol.  aminovalerianic  acid,  and  2  mol.  a-proline. 
Scombrine  contains  only  arginine,  alanine,  and  a-proline.  The  following 
summary  according  to  Kossel  ^  gives  a  view  of  the  cleavage  products  of 
the  protamines  thus  far  investigated. 


Alanine 

iiJerine 

Aminovalerianic  acid 

Leucine 

Arginine 

Lysine 

Histidine 

«- Proline 

Tyrosine  • 

Tryptophane 


Scom- 

Salmine. 

Clupeine. 

Sturine. 

Cyclop- 

a-Cyp- 

brine. 

terine. 

rinine. 

+ 

0 

+ 

+ 

? 

? 

0 

+ 

+ 

0 

? 

? 

0 

+ 

+ 

0 

? 

+ 

0 

0 

0 

+ 

? 

•? 

+ 

+ 

+ 

+ 

+ 

+ 

0 

0 

0 

+ 

0 

+ 

0 

0 

0 

+ 

0 

0 

+ 

+ 

+ 

c 

? 

9 

0 

0 

0 

0 

+ 

0 

0 

0 

0 

0 

+ 

0 

/J-Cyp- 
rinine. 


Solutions  of  these  bases  in  water  are  alkaline  and  have  the  pro]3erty 
of  giving  precipitates  with  ammoniacal  solutions  of  proteins  or  primary  pro- 
teoses. These  precipitates  are  considered  as  hist  ones  by  Kossel.  The  salts 
with  mineral  acids  are  soluble  in  water,  but  insoluble  in  alcohol  and 
ether.  They  are  more  or  less  readily  precipitated  by  neutral  salts  (NaCl). 
Among  the  salts  of  the  protamines,  the  sulphate,  picrate,  and  the  double- 
platinum  chloride  are  the  most  important  and  are  used  in  the  preparation 
of  the  protamines.  The  protamines  are,  like  the  proteins,  levog3'rate.  They 
give  the  biuret  test  beautifully,  but  with  the  exception  of  cyclopterine  and 
_,i9-c3'prinine  do  not  give  Millon's  reaction.  The  protamine  salts  are  pre- 
cipitated in  neutral  or  even  faintly  alkaline  solutions  by  phosphotungstic 
acid,  picric  acid,  chromic  acid,  and  alkali  ferrocyanides. 

The  protamines  are  prepared,  according  to  Kossel,  by  extracting  the 
heads  of  the  spermatozoa,  which  have  previously  been  extracted  with 
alcohol  and  ether,  with  dilute  sulphuric  acid  (1-2  per  cent),  filtering,  and 
precipitating  with  4  vols,  of  alcohol.  The  sulphate  may  be  purified  by 
repeated  solution  in  water  and  precipitation  with  alcohol,  and  if  necessary, 
conversion  into  the  ]:)icrate.  For  more  details  see  the  works  of  Kossel. 
The  double-platinum  salt  is  best  suited  for  analysis  and  can  be  obtained, 
according  to  Goto,  by  j^recipitating  the  methyl-alcohol  solution  of  the 
protamine  hydrochloride  with  platinum  chloride.  Miescher  also  precipi- 
tates the  base  as  a  douLle-platinum  salt. 


'  Zeitschr.  f.  }  hy.sio).  Chcni.,  44. 


COMPOUND  PROTEIDS.  65 


II.  Compound  Proteids. 

With  this  name  we  designate  a  class  of  bodies  which  are  more  complex 
than  the  proteids,  and  which  yield  as  primary  splitting  products  proteids 
on  the  one  side  and  non-proteid  bodies,  such  as  pigments,  carbohydrates, 
nucleic  acids,  etc.,  on  the  other.^ 

The  compound  proteids  known  at  the  present  time  are  di\-ided  into 
three  chief  groups.  These  are  the  hcemoglobins,  the  glucoproteids,  and  the 
nudeoproteids.  The  haemoglobins  will  be  discussed  in  a  following  chapter 
(Chapter  YI,  on  the  blood) . 

Glucoproteids  are  those  compound  proteids  which  on  decomposition 
yield  a  proteid  on  the  one  side,  and  a  carbohydrate  or  derivatives  of  the 
same  on  the  other,  but  no  purine  bodies.  Some  glucoproteids  are  free  from 
phosphorus  (mucin  substances,  chondroproteids,  and  hyalogens),  and 
some  contain  phosphorus  (phosphoglucoproteids). 

The  glucoproteids  free  from  phosphorus  may,  as  regards  the  nature  of 
the  carbohydrate  groups  split  off,  be  divided  into  two  chief  groups,  the 
mucin  substances  and  the  chondroproteids.  The  first  yield  on  hydrolytic 
cleavage  an  amino-sugar,  which  has  been  shown  to  be  glucosamine  in  all 
cases  except  one.-  In  the  chondroproteids,  on  the  contrary,  the  proteid 
is  united  to  chondroit in-sulphuric  acid. 

Mucin  Substances.  These  bodies  contain  carbon,  hydrogen,  nitrogen, 
sulphur,  and  oxygen.  Compared  with  proteids  they  are  poorer  in  nitrogen 
and  as  a  rule  have  also  considerably  less  carbon.  The  carbohydrate  complex, 
M^hose  nature  has  been  showm  by  the  investigations  of  Fr.  MiJLLER  ^  and 
his  pupils,  occurs,  as  it  seems,  in  the  mucin  substances  as  a  pol3'saccharide 
related  to  chitosan,  which  on  hydrolytic  cleavage  yields  glucosamine 
(chitosamine),  and,  at  least  in  most  cases,  also  acetic  acid.  The  mucin 
substances  differ  ver\^  markedly  among  one  another,  hence  we  divide  them 
into  two  groups,  the  mucins  and  the  mucoids. 

The  true  yyiucins  are  characterized  by  the  fact  that  their  natural  solu- 
tions, or  solutions  prepared  by  the  aid  of  a  trace  of  alkali,  are  mucilaginous, 
ropy,  and  give  a  precipitate  with  acetic  acid  which  is  insoluble  in  excess  of 
acid  or  soluble  only  with  great  difficult3^     The  mucoids  do  not  show  these 

'  Hoppe-S.eyler  has  given  the  name  prote'ide  to  these  compound  proteids,  but  as 
this  term  is  misleading  in  EngHsh  we  do  not  use  it  in  English  classifications  in  this 
sense. 

^  See  Schulz  and  Ditthorn,  Zeitschr.  f.  physiol.  Chem.,  29.  When  both  carbo- 
hydrate groups  are  simultaneously  combined  with  one  body,  then  probably  we  are  not 
dealing  with  a  chemical  individual,  but  rather  with  a  mixture. 

^  See  Fr.  Miiller,  Zeitschr.  f.  Biologie,  42,  which  contains  all  the  pertinent  litera- 
ture, and  also  L.  Langstein,  Die  Bildung  von  Kohlenhydraten  aus  Eiweiss,  Ergebnisse 
der  Physiologie,  Jahrg.  I,  Abt,  1. 


66  THE  PROTEIN  SUBSTANCES. 

physical  properties  and  have  other  soIubiUties  and  precipitation  properties. 
As  we  have  intermediate  steps  between  different  protein  bodies,  so  also 
we  have  such  between  true  mucins  and  mucoids,  and  a  sharp  line  cannot 
be  drawn  between  these  two  groups. 

It  is  just  as  difficult  at  present  to  draw  a  sharp  line  between  the  pro- 
teids  and  the  mucins  or  mucoids,  since  we  have  been  able  to  split  off  carbo- 
hydrate complexes  from  several  proteids,  and  the  proteids  of  the  white 
of  egg  are  undoubtedly  glucoproteids.  It  is  immaterial  whether  we  con- 
sider these  glucoproteids  as  belonging  to  the  mucoids  or  to  a  special  group. 
From  a  comparative  chemical  standpoint,  they  undoubtedl}'  belong  to  the 
mucoid  group,  representatives  of  which  occur  in  eggs  to  a  considerable 
extent. 

True  mucins  are  secreted  by  the  larger  mucous  glands,  by  certain  mucous 
membranes,  and  by  the  skin  of  snails  and  other  animals.  True  mucin  also 
occurs  in  the  navel-cord.  Sometimes,  as  in  snails  and  in  the  membrane 
of  the  frog-egg  (Giacosa  i),  a  mother-substance  of  mucin,  a  mucinogen, 
has  been  found  which  may  be  converted  into  mucin  by  alkalies.  I\Iucoid 
substances  are  found  in  certain  cysts,  in  the  cornea,  the  crystalline  lens, 
white  of  egg,  and  in  certain  ascitic  fluids.  The  so-called  tendon-mucin, 
which,  according  to  the  recent  investigations  of  Levexe  and  of  Cutter,  and 
GiES,2  contains  chondroitin-sulphuric  acid  or  a  related  substance,  cannot 
be  classified  as  a  mucin,  but  must,  like  the  chondromucoid  and  the  osseo- 
mucoid, be  classified  as  chondroproteid.  As  the  mucin  question  has  not 
been  sufficiently  studied,  it  is  at  the  present  time  impossible  to  give  any 
positive  statements  in  regard  to  the  occurrence  of  mucins  and  mucoids, 
especially  as  without  doubt  in  many  cases  non-mucinous  sul^stances  have 
been  described  as  mucins. 

I.  True  Mucins.  Thus  far  we  have  been  able  to  obtain  only  a  few 
mucins  in  a  pure  and  unchanged  condition,  because  of  the  reagents  used. 
The  elementary  analyses  of  these  mucins  have  given  the  following  results: 

c           H  N           s 
Mucin    from    mucous    membrane    (air- 
passages) 48.26  6.91  10.7  1.4  (Fr.  Muli.er) 

Mucin  from  submaxillary 48.84  6.80  12.32  0.84  (H.\mm.\rsten*) 

Mucin  from  snail 50.32  6.84  13.65  1 .75  (Hammarstex^) 

Synovial  mucin 51 .05  6.53  13.01  1 .34  (v.  Holst  ") 

MtJLLER  obtained  35  per  cent  glucosamine  from  mucous-membrane 
mucin  and  23.5  per  cent  from  the  submaxillary  mucin. 

'Giacosa,  Zeitschr  f  physiol.  Chem.  7;  Hammarsten,  Pflliger's  Archiv,  30,  and 
Skand.  Arch.  f.  Physiol.,  17. 

2  Levene,  Zeitschr.  f.  physiol.  Chem.   31;  Cutter  and  Gies,  Amer.  Journ.  of  Physiol.,  6. 

3  Fr.  Miiller,  Zeitschr.  f.  Biologic,  42;  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  12, 
and  Pfluger's  Arch.,  3G. 

*  Zeitschr.  f.  phy,?iol  Chem.,  43. 


MUCINS.  67 

By  the  action  of  superheated  steam  on  mucin  a  carbohydrate,  animal 
gum  (Landwehp",  is  split  off.  This  has  not  been  substantiated  by  other 
investigators,  such  as  Folin  and  Fr.  iMtJLLER.^  Instead  of  a  non-nitrogenous 
gum  a  nitrogenous  carbohydrate  derivative  was  always  obtained. 

On  boiling  mucin  with  dilute  mineral  acids,  acid  albuminate  and  bodies 
similar  to  proteoses  are  obtained,  besides  a  reducing  substance  which  is 
not  free  glucosamine  (Steudel^).  By  the  action  of  strong  acids  upon 
mucins  or  mucoids  Otori  ^  obtained  several  of  the  cleavage  products  of 
the  proteins,  such  as  leucine,  tyrosine,  glycocoll,  glutamic  acid,  oxalic  acid, 
guanidine,  arginine,  lysine,  and  humus  substances,  and  also  carbohydrate 
cleavage  products,  such  as  levulinic  acid.  Certain  mucins,  as  the  submaxil- 
lary mucin,  are  easily  changed  by  very  dilute  alkalies,  as  lime-water,  while 
others,  such  as  tendon-mucin,  are  not  affected.  If  a  strong  caustic-alkali 
solution,  such  as  5  per  cent  KOH  solution,  is  allowed  to  act  on  submaxillary 
mucin,  we  obtain  alkali  albuminate,  bodies  similar  to  proteoses  and  peptones 
and  one  or  more  substances  of  an  acid  reaction  and  with  strong  reducing 
powers. 

On  peptic  digestion  proteoses  and  peptone-like  bodies,  still  containing 
the  carbohydrate  group,  are  produced.  On  tryptic  digestion  still  simpler 
cleavage  products  are  formed,  namely,  leucine,  tyrosine,  and  tryptophane 
(PosNER  and  Gies*).  The  glucosamine,  so  far  as  we  know,  is  not  split  off 
by  proteolytic  enzymes,  but  only  after  strong  hydrolysis  with  acids,  and 
this  speaks  against  the  assumption  that  the  glucosamine  group  exists 
as   a  glucoside-like  combination   in  the  mucin   molecule   (Neuberg  and 

MlLCHNER^). 

In  one  or  another  respect  the  various  mucins  act  somewhat  dissimilarly. 
For  example,  the  snail  and  sputum  mucins  are  insoluble  in  dilute  hydro- 
chloric acid  of  1-2  p.  m.,  while  the  mucin  of  the  submaxillary  gland  and 
the  navel-cord  is  soluble.  The  former  become  flaky  with  acetic  acid,  while 
the  submaxillary  mucin  is  precipitated  in  more  or  less  fibrous,  tough  masses. 
Still  all  the  mucins  have  certain  reactions  in  common. 

In  the  drj^  state  mucin  forms  a  white  or  yellowish-gray  powder.  When 
moist  it  forms,  on  the  contrary,  flakes  or  yellowish-white  tough  lumps  or 
masses.  The  mucins  are  acid  in  reaction.  They  give  the  color  reactions  of 
the  proteins.  They  are  not  soluble  in  water,  but  may  give  a  neutral  solu- 
tion with  water  with  the  aid  of  small  amounts  of  alkali.     Such  a  solution 


'  Landwehr,  Zeitschr.  f.  physiol.  Chem.,  8,  9;  also  Pfliiger's  Arch.,  39  and  40; 
Folin,  Zeitschr.  f.  physiol.  Chem.,  23;  Fr.  Miiller,  Sitzungsber.  d.  Gesellsch.  zur  Beford. 
d.  gesammt.  Naturwiss.  zu  Marburg,  1896. 

'  Zeitschr.  f.  physiol.  Chem.,  34. 

Ubul.,  42  and  43. 

*Amer.  Journ.  of  Physiol.,  11. 

*  Berl.  klin.  Wochenschr.,  1904. 


68  THE  PROTEIN  SUBSTANCES. 

does  not  coagulate  on  boiling,  but  acetic  acid  gives  at  the  normal  tempera- 
ture a  precipitate  which  is  nearly  insoluble  in  an  excess  of  the  precipitant. 
If  5-10  per  cent  NaCl  be  added  to  a  mucin  solution,  this  can  now  be  care- 
fully acidified  with  acetic  acid  without  giving  a  precipitate.  Such  acidified 
solutions  are  copiously  precipitated  by  tannic  acid;  with  potassium  ferro- 
cyanide  they  give  no  precipitate,  but  on  sufficient  concentration  they 
become  thick  or  viscous.  A  neutral  solution  of  alkali  mucin  is  precipitated 
by  alcohol  in  the  presence  of  neutral  salts;  it  is  also  precipitated  by  several 
metallic  salts.  If  mucin  is  heated  on  the  water-bath  with  dilute  hydro- 
chloric acid  of  about  2  per  cent,  the  liquid  gradually  becomes  a  yellowish 
or  dark  brown  and  reduces  copper  salts  in  alkaline  solutions. 

The  mucin  most  readily  obtained  in  large  quantities  is  the  submaxillary 
mucin,  which  may  be  prepared  in  the  following  way:  The  filtered  watery 
extract  of  the  gland,  free  from  form-elements  and  as  colorless  as  possible, 
is  treated  with  25  per  cent  hydrochloric  acid,  so  that  the  liquid  contains 
1.5  p.  m.  HCl.  On  the  addition  of  the  acid  the  mucin  is  immediately  pre- 
cipitated, but  dissolves  on  stirring.  If  this  acid  liquid  is  immediately 
diluted  with  2-3  vols,  of  water,  the  mucin  separates  and  may  be  purified 
by  redissolving  in  1-5  p.  m.  acid,  and  diluting  with  water  and  washing 
therewith.  The  mucin  of  the  navel-cord  may  be  prepared  in  the  same  way. 
As  a  rule  the  mucins  can  be  prepared  by  precipitation  with  acetic  acid  and 
repeated  solution  in  dilute  lime-water  or  alkali  and  reprecipitation  with 
acetic  acid.  Finally  they  are  treated  with  alcohol  and  ether.  In  the 
preparation  of  sputum  mucin  a  very  complicated  method  is  necessary  (Fr. 

MtJLLER). 

The  precipitation  by  acetic  acid,  as  shown  by  Hammarsten,^  is  not  applicable 
in  the  preparation  of  submaxillary  mucin,  because  another  proteid  substance  is 
precipitated  with  the  mucin,  but  remains  in  solution  on  using  the  hydrochloric- 
acid  method  above  described.  Posner  and  Gies  ^  have  by  special  experiments 
shown  the  power  of  mucins  of  precipitating  proteids,  and  this  makes  the  ordi- 
nary method  of  precipitating  with  acetic  acid  questionable. 

2.  Mucoids  or  Mucinoids.  In  this  group  we  must  include  those  non- 
phosphorized  glucoproteids  which  are  neither  true  mucins  nor  chondro- 
proteids,  even  though  they  show  amongst  themselves  such  differences  in 
behavior  that  they  can  be  divided  into  several  subgroups  of  mucoids. 
To  the  mucoids  belong  pseiidomucin,  the  probably  related  body  colloid, 
ovomucoid,  and  other  bodies,  which  on  account  of  their  differences  will  be 
best  treated  individually  in  their  respective  chapters. 

Hyalogens.  Under  this  name  Krukenberg  ^  has  designated  a  number  of 
differing  bodies,  which  are  characterized  by  the  following:  By  the  action  of 
alkalies  they  change,  with  the  splitting  off  of  sulphur  and  some  nitrogen,  into 
soluble  nitrogenized  products  called  by  him  hyalines  and  which  yield  a  pure  car- 

'  Zeitschr.  f.  physiol.  Chem.,  12. 

*  Amer.  Journ.  of  Physiol.,  11. 

3  Verh.  d.  physik.-med.  Gesellsch.  zu  Wiirzburg,  1883;  also  Zeitschr.  f.  Biologie,  22. 


AMYLOID.  69 

bohydrate  by  further  decomposition.  We  find  that  very  heterogeneous  substances 
are  included  in  this  group.  Certain  of  these  hyalogens  seem  undoubtedly  to 
be  glucoproteids.  Neossin  ^  of  the  Chinese  edible  swallow 's-nest,  mernbranin  ^ 
of  Descemet's  membrane  and  of  the  capsule  of  the  crystalline  lens,  and  spiro- 
'jraphin  ^  of  the  skeletal  tissue  of  the  worm  Spirograplais  seem  to  act  as  such. 
Others  on  the  contrary,  such  as  hyalin  *  of  the  walls  of  hydatid  cysts,  and  onu- 
phin^  from  the  tubes  of  Onuphis  tubicola,  do  not  seem  to  be  compound  proteids. 
The  so-called  mvcin  of  the  holothurcs  ^  and  chondrosin  ^  of  the  sponge,  Chondrosia 
reniformis,  and  others  may  also  be  classed  with  the  hyalogens.  As  the  various 
bodies  designated  by  Krukenberg  as  hyalogens  are  very  dissimilar,  it  is  not 
of  much  advantage  to  arrange  these  in  special  groups. 

3.  Chondroproteids  are  those  glucoproteids  which  as  primary  cleavage 
products  yield  proteid  and  an  ethereal  sulphuric  acid  containing  a  carbo- 
hydrate, chondroitin-suliphuric  acid.  Chondromucoid ,  occurring  in  cartilage, 
is  the  best  example  of  this  group.  Amyloid  occurring  under  pathological 
conditions  also  belongs  to  this  group.  On  account  of  the  property  of  chon- 
droitin-sulphuric  acid  of  precipitating  proteid,  it  is  also  possible  that  under 
certain  circumstances  combinations  of  this  acid  with  proteid  may  be  pre- 
cipitated from  the  urine  and  be  considered  as  chondroproteids. 

The  chondromucoid,  the  so-called  tendon-mucin,  and  the  osseomucoid 
have  greatest  interest  as  constituents  of  cartilage,  of  the  connective  tissues, 
and  of  the  bones,  and  on  this  account  these  bodies  and  their  cleavage  prod- 
uct, chondroit in-sulphuric  acid,  will  be  treated  in  a  following  chapter  (X). 
On  the  contrary,  amyloid,  which  has  alwaj^s  been  considered  in  connection 
with  the  protein  substances,  will  be  described  here. 

Amyloid,  so  called  by  Virchow,  is  a  protein  substance  appearing  under 
pathological  conditions  in  the  internal  organs,  such  as  the  spleen,  liver,  and 
kidneys,  as  infiltrations;  and  in  serous  membranes  as  granules  with  con- 
centric la^^ers.  It  probably  also  occurs  as  a  constituent  of  certain  prostate 
calculi.  The  chondroproteid  occurring  under  physiological  conditions  in 
the  Avails  of  the  arteries  is  perhaps,  according  to  Krawkow,  very  nearly 
related  to  the  amyloid  substance,  but  not  identical  with  it,  as  shown  by 
Neuberg.^ 

The  amyloid  prepared  by  Krawkow  and  Neuberg  had  about  the  same 
composition:  C  49.0-50.1;  H  7-7.2;  N  14-14.1,  and  S  1.8-2.S  per  cent. 
The  aorta  amyloid  of  man  and  of  the  horse  contained  respectively  C  49.6 

'  Krukenberg,  Zeitschr,  f,  Biologie,  22. 

'  C.  Th.  Morner,  Zeitschr.  f.  physiol.  Chem.,  IS. 

'Krukenberg,  Wiirzburg,  Verhandl.  1883;   also  Zeitschr.  f.  Biologie,  22. 

*A.  Liicke,  Virchow's  Arch.,  19;  also  Krukenberg,  Vergleichende  physiol.  Stud., 
Series  1  and  2,  1881. 

^  Schmiedeberg,  Mitth.  aus  d.  zool.  Stat,  zu  Neapel,  3,  1882. 

^  Hilger,  Pffiiger's  Archiv,  3. 

'  Krukenberg,  Zeitschr.  f.  Biologie,  22. 

"  Krawkow,  Arch.  f.  exp.  Path.  u.  Pharm.,  40,  which  contains  the  literature;  Neu- 
berg, Verhandl.  d.  d.  Pathol.  Gesellsch.  1904. 


70  THE  PROTEIN  SUBSTANCES. 

and  5U.5;  H  7.2;  N  14.4  and  13.8;  S  2.3  and  2.5  per  cent.  According  to 
Neuberg,  aorta  amyloid  differs  from  spleen  and  liver  amyloid  by  a  different 
division  of  the  nitrogen,  which  is  evident  from  the  follo\Aing: 

Monamino-X  Diamino-N  Amide-N 

Liver  amyloid 43 .2                      ol .  2  4.9 

Spleen  amyloid 30. 6                      57.  U  11.2 

Aorta  amyloid 54.9                     36.0  8.8 

From  liver  amyloid  Neuberg  obtained  glycocoll  0.8;  leucine  22.2;  glu- 
tamic acid  3.8;  tyrosine  4.0;  a-proline  3.1;  arginine  13.0,  and  lysine 
11.6  per  cent. 

By  the  action  of  alkali,  amyloid  splits  into  protein  and  chondroitin- 
sulphuric  acid  (see  Chapter  X),  and  according  to  Krawkow  it  is  there- 
fore a  firm,  perhaps  ester-like  combination  of  this  acid  with  protein. 
The  protein,  from  the  investigations  of  Neuberg,  is  of  a  basic  nature  and 
most  comparable  to  the  histones.  According  to  Neuberg,  amyloid  is  a 
transformation  product  of  the  proteins,  just  as  are  the  protamines,  and  the 
differences  between  liver,  spleen,  and  aorta  amyloid  indicate  various  phases 
of  this  transformation. 

Amyloid  is  an  amorphous  white  substance,  insoluble  in  water,  alcohol, 
ether,  dilute  hydrochloric  and  acetic  acids.  It  is  soluble  in  concentrated 
hydrochloric  acid  or  caustic  alkali  with  decomposition.  On  boiling  with 
dilute  hydrochloric  acid  it  yields  sulphuric  acid  and  a  reducing  substance. 
It  is  not  dissolved  by  gastric  juice,  according  to  Krawkow  and  in  agree- 
ment with  most  of  the  older  statements.  It  is  nevertheless  changed  so 
that  it  is  soluble  in  dilute  ammonia,  while  the  typical  amyloid  is  insoluble 
therein.  Neuberg  finds  on  the  contrary  that  amyloid  (from  liver)  is 
digested  by  pepsin  as  well  as  by  trj^psin,  although  more  slowly  than  fibrin, 
and  that  it  is  also  destroyed  in  autolysis,  so  that  in  life  an  absorption 
is  possible.  Amyloid  gives  the  xanthoproteic  reaction  and  the  reactions 
of  MiLLOx  and  Adamkiewicz.  Its  most  important  property  is  its  behavior 
with  certain  coloring  matters.  It  is  colored  reddish  brown  or  a  dingy 
violet  by  iodine;  a  violet  or  blue  by  iodine  and  sulphuric  acid;  red  by 
methylaniline  iodide,  especially  on  the  addition  of  acetic  acid;  and  red 
also  by  aniline  green.  Of  these  color  reactions  those  with  aniline  dyes 
are  the  most  important.  The  iodine  reaction  appears  less  constant  and 
is  greatly  dependent  upon  the  physical  condition  of  the  amyloid.  The 
color  reactions  are  due  to  the  presence  of  the  chondroitin-sulphuric  acid 
component. 

The  preparation  of  amyloid  may  be  performed  as  follows  according 
to  Modrzejewski  and  Krawkow.^  The  finely  divided  organ  is  exhausted 
first  with  water  and  then  with  dilute  ammonia,  which  leaves  the  insoluble 

'  Modrzejewski,  Arch.  f.  exp.  Path.  u.  Pharm.,  1;   Krawkow,  I.  c. 


NUCLEOPROTEIDS.  71 

amyloid  and  removes  the  free  or  the  combined  chondroit in-sulphuric  acid, 
besides  other  substances.  The  product,  after  being  washed  with  water,  ia 
digested  ^^ith  pepsin  for  several  days  at  38°  C.  The  residue,  after  washins 
"with  hydrochloric  acid  and  water,  is  dissolved  in  dilute  ammonia,  filtered, 
again  precipitated  with  dilute  hydrochloric  acid,  dissolved,  if  necessary, 
in  ammonia,  precipitated  a  second  time  with  hydrochloric  acid,  washed 
with  water,  the  precipitate  dissolved  in  bar\'ta-water,  which  leaves  the 
nucleins  undissolved,  and  the  barium  filtrate  precipitated  "^nth  hydrochloric 
acid,  and  then  washed  with  water,  alcohol,  and  ether. 

Phosphoglucoproteids.  This  group  includes  the  phosphorized  glucoproteids. 
They  yield  no  xanthine  substances  (nuclein  bases)  as  cleavage  products.  They 
are  not  nucleoproteids  and  therefore  they  must  not  be  considered  together 
with  the  gluconucleoproteids  (nucleoglucoproteids;  or  mistaken  for  them.  On 
pepsin  digestion  they  ma}',  like  certain  nucleoalbumins,  yield  pseudonuclein, 
but  they  differ  from  the  nucleoalbumins  in  that  they  yield  a  reducing  substance 
on  boiling  with  dilute  acid.  They  differ  from  the  gluconucleoproteids  in  that 
they  do  not,  as  above  mentioned,  yield  any  xanthine  bodies. 

Only  two  phosphorized  glucoproteids  are  known  at  the  present  time,  namely, 
ichthidin,  occurring  in  carp  eggs  and  studied  by  Walter  ^  and  which  was  con- 
sidered as  a  vitellin  for  a  time.  Ichthulin  has  the  following  composition:  C  .53.52; 
H  7.71;  X  15.64;  S  0.41;  P  0.4.3;  Fe  0.10  per  cent.  In  regard  to  solubilities  it 
is  similar  to  a  globulin.  Walter  has  prepared  a  reducing  substance  from  the 
paranuclein  of  ichthulin  which  gave  a  highly  crystalline  compound  with  phenyl- 
hydrazine. 

Another  phosphoglucoproteid  is  helicoproteid,  obtained  by  Hammarsten  - 
from  the  glands  of  the  snail  Helix  pomatia.  It  has  the  following  composition: 
C  46.99 ;  H  6.78 ;  N  6.08 ;  S  0.62 ;  P  0.47  per  cent.  It  is  converted  into  a  gumn.y, 
levorotatory  carbohydrate,  called  atnmal  sinistrin,  by  the  action  of  alkalies. 
On  boiling  with  an  acid  it  yields  a  dextrorotatory  reducing  substance. 

The  compound  proteid  found  by  Schulz  and  Ditthorx  ^  in  the  spawn 
of  the  frog  probably  belongs  to  this  group,  but  instead  of  glucosamine  it 
gives  galactosamine  on  cleavage. 

Nucleoproteids.  With  this  name  we  designate  those  compound  pro- 
teids  which  yield  true  nucleins  (see  Chapter  V)  on  pepsin  digestion,  and 
on  cleavage  ^^•ith  dilute  caustic  alkali  yield  proteid  and  nucleic  acid. 

The  nucleoproteids  seem  to  be  widely  diffused  in  the  animal  body.  They 
occur  chiefly  in  the  cell-nuclei,  but  they  also  often  occur  in  the  proto- 
plasm. They  may  pass  into  the  animal  fluids  on  the  destruction  of  the 
cells,  hence  nucleoproteids  have  also  been  found  in  blood-serum  and  other 
fluids. 

They  may  be  considered  as  combinations  of  a  proteid  nucleus  with  a 
side  chain,  which  Kossel  calls  the  prcsthetic  group.  This  side  chain, 
which  contains  the  phosphorus,  may  be  split  off  as  nucleic  acid  (see  Chapter 
Y)  on  treatment  with  alkali.  As  we  have  several  nucleic  acids,  it  follows 
that  we  must  have  different  nucleoproteids,  depending  upon  the  nucleic  acid 

'  Zeitschr.  f.  physiol.  Chem.,  l.>. 
*  Hammarsten,  Pfliiger's  Arch.,  3B. 
'  Zeitschr.  f .  physiol.  Chem.,  23. 


72  THE  PROTEIN  SUBSTANCES. 

united  with  the  proteid.  Certain  nucleic  acids  contain  a  readily  split-off 
sugar  (pentose  or  hexose);  others,  on  the  contrar}^  do  not.  In  the  first 
case  we  obtain  from  the  corresponding  nucleoproteid  a  reducing  sugar 
on  boiling  with  dilute  mineral  acid,  while  in  the  other  case  this  is  not  pos- 
sible. Corresponding  to  this  different  behavior  we  may  divide  off  a  special 
group  of  nucleoproteids,  the  gluconucleoproteids  or  nucleoglucoproteids. 
Such  gluconucleoproteids,  yielding  pentoses,  occur  in  yeast-cells,  and,  as  it 
appears,  are  widely  distributed  in  the  animal  organism  (Blumenthal, 
Gruxd  1). 

The  native  nucleoproteids  contain  a  variable  but  not  a  high  percen- 
tage of  phosphorus,  which  Halliburton  2  found  to  vary  between  0.5  per 
cent  and  1.6  per  cent.  On  heating  their  solutions,  as  well  as  by  the  action 
of  dilute  acids,  a  modification  of  the  compound  proteid  takes  place,  and 
nucleoproteids  of  strong  acid  character,  poorer  in  proteid  but  richer  in 
phosphoi-us,  are  formed.  The  native  nucleoproteids  have  faint  acid  proper- 
ties and  are  insoluble  in  water,  but  their  alkali  compounds,  which  are 
soluble  in  water,  split  on  heating  their  solutions  into  coagulated  proteid  and 
a  nucleoproteid  rich  in  phosphorus,  which  remains  in  solution.  In  peptic 
digestion  they  yield  so-called  true  nuclein,  which  is  also  a  nucleoprotjeid 
poor  in  proteid.  The  proteid  can  be  precipitated  by  acetic  acid  from  its 
alkali  compound,  and  the  precipitate  dissolves  with  more  or  less  readiness 
in  an  excess  of  the  acid.  A  confusion  may  occur  here  with  nucleoalbumins 
and  also  with  mucin  substances.  This  confusion  may  be  avoided  by 
warming  the  ])ody  for  some  time  on  the  water-bath  with  dilute  sulphuric 
acid,  nearly  neutralizing  the  boiling-hot  fluid  with  barium  hydrate,  filtering 
as  quickly  as  possible  while  boiling  hot,  and  testing  the  filtrate  for  purine 
bodies  with  copper  sulphate  and  bisulphite  according  to  the  method  given 
on  page  163.  Any  precipitate  formed  is  examined  more  closely  by  the 
method  there  given.  The  nucleoproteids  give  the  color  reactions  of  the 
proteins,  but  those  which  have  been  investigated  are  dextrorotatory^  and 
not  levorotatory  (Gamgee  and  Jones  3). 

The  properties  of  the  various  nucleoproteids  are  given  in  detail  in  the 
various  chapters  which  follow. 

III.  Albuminoids  or  Proteinoids. 

Under  this  name  we  collect  into  a  special  group  all  those  protein  bodies 
which  cannot  be  placed  in  either  of  the  other  two  groups,  although  they 
differ  essentially  among  themselves,  and  from  a  chemical  standpoint  do 

'  Blumenthal,  Berlin,  klin.  Wochenschr.,  1897,  and  Zeitschr.  f,  klin,  Med.,  34; 
Grund,  Zeitschr.  f.  physiol.  Chem.,  35.  See  also  Bendix  and  Ebstein,  Zeitschr.  f. 
allgem.  Phys.,  2;   Levene  and  Mandel,  Zeitschr.  f.  physiol.  Chem.,  47. 

^  Journ.  of  Physiol.,  IS. 

^  Hofmeister's  Beitrage,  4. 


KERATINS.  73 

not  show  any  radical  difference  from  the  true  protein  bodies.  The  most 
important  and  abundant  of  the  bodies  belonging  to  this  group  are  important 
constituents  of  the  animal  skeleton  or  the  cutaneous  structure.  They  occur 
as  a  rule  in  an  insoluble  state  in  the  organism,  and  they  are  distinguished 
in  most  cases  by  a  pronounced  resistance  to  reagents  which  dissolve  proteins 
or  to  chemical  reagents  in  general. 

The  Keratin  Group.  Keratin  is  the  chief  constituent  of  the  homy 
structure,  of  the  epidermis,  of  hair,  wool,  of  the  nails,  hoofs,  horns,  feathers,, 
of  tortoise-shell,  etc.,  etc.  Keratin  is  also  found  as  neurokeratin  (Kuhxe) 
in  the  brain  and  nerves.  The  shell-membrane  of  the  hen's  egg  seems 
also  to  consist  of  keratin,  and  according  to  NeumeisterI  the  organic 
matrix  of  the  egg-shells  of  various  vertebrate  animals  belongs  in  most 
cases  to  the  keratin  group. 

It  seems  that  there  exist  a  number  of  keratins,  and  these  form  a  special 
group  of  bodies.  This  fact,  together  with  the  difficulty  in  isolating  the 
keratin  from  the  tissues  in  a  pure  condition  ^\ithout  a  partial  decomposi- 
tion, is  sufficient  explanation  for  the  variation  in  the  elementary  composi- 
tion given  below.  As  examples  the  analyses  of  a  few  tissues  rich  in  keratin 
and  of  keratins  are  given.^ 

c  H                   N  s            0 

Human  hair.  ..  .  50.65  6.36  17.14  5.00  20 . 85  (v.  L.var) 

Nail 51.00  6.94  17.51  2.80  21.75  (Mulder) 

Neurokeratin  .  .  .  .56.11-58.45  7.26-8.02  11.46-14.32  1.63-2.24 (Kuhne) 

Horn  (average)..  50.86  6.94  3.20  (Horbaczewski) 

Tortoise-i^hell.  .  .  54.89  6.56  16.77  2.22  19.56   (Mulder) 

Shell-membrane.  49.78  6.94  16.43  4.25  22.90   (Lindvall) 

!MoHR  2  has  determined  the  quantity  of  sulphur  in  various  keratin  sub- 
stances. Sulphur  is  in  great  part  in  loose  combination,  and  it  is  chiefly 
removed  by  the  action  of  alkalies  (as  sulphides),  or  indeed  in  part  by  boiling 
with  water.  Combs  of  lead  after  long  usage  become  black,  and  this  is  due 
to  the  action  of  the  sulphur  of  the  hair.  On  heating  keratin  with  water  in 
sealed  tubes  to  a  temperature  of  150°  C.  or  higher,  it  dissolves,  with  the 
elimination  of  sulphuretted  hydrogen  or  mercaptan  (Bauer),  and  the 
solution  contains  proteose-like  substances  (Krukenberg)  called  atmidkera- 
tin  and  atmidkeratose  by  Bauer.^  Keratin  is  dissolved  by  alkalies,  especially 
on  warming,  producing  besides  alkali  sulphides  also  proteose  substances. 

>  Kiihne  and  Ewald,  Verh.  d.  naturhistor.-med.  Vereins  zu  Heidelberg  (N.  F.),  1; 
also  Kiihne  and  Chittenden,  Zeitschr.  f.  Biologic,  2(>;    Neumeister,  ibid.,  31. 

^  V.  Laar,  Annal.  d.  Chem.  u.  Pharm.,  4o;  Mulder,  Versuch  einer  allgem.  physioL 
Chem.,  Braunschweig,  1844-51;  Kiihne,  Zeitschr.  f.  Biologic,  20;  Horbaczewski,  see 
Drechsel  in  Ladenburg's  Handworterbuch  d..  Chem.,  3;  Lindvall,  Maly's  Jahres- 
bericht,  1881. 

^Zeitschr.  f.  physiol.  Chem..  20. 

*  Krukenberg,  Untersuch.  iiber  d.  chem.  Bau  d.  Eiweisskorper,  Sitzungsber.  d. 


74  THE  PROTEIN  SUBSTANCES. 

Besides  the  well-known  cleavage  products  such  as  leucine,  tyrosine, 
aspartic  acid,  glutamic  acid,  arginine,  and  lysine,  Fischer  and  Dorping- 
HAUS  ^  have  recently  found  glycocoll,  alanine,  a-aminovalerianic  acid, 
^-proline,  serine,  phenylalanine,  and  pyrrolidone-carboxylic  acid  (secondary 
from  glutamic  acid)  among  the  cleavage  products  of  horn  substances. 
Emmerling  claims  to  have  found  cystine  as  a  sulphurized  cleavage  product, 
but  K.  Morner2  was  the  first  to  positively  prove  the  abundant  occur- 
rence of  cystine  in  the  cleavage  products.  Morner  obtained  from  ox-horn, 
human  hair,  and  the  shell-membrane  of  the  hen's  egg  6.8,  13.92,  and  7.62 
per  cent  cystine  calculated  on  the  basis  of  the  dry  substance.  From  the 
amount  of  sulphur  split  off  by  alkali,  he  concludes  that,  at  least  in  ox-horn 
and  human  hair,  all  the  sulphur  exists  as  cystine.  Galimard  ^  was  able 
to  get  only  a  qualitative  test  for  cystine  in  the  keratin  of  the  adder  eggs. 
SuTER,  Morner,  and  Friedmann*  have  obtained  a-thiolactic  acid  as  a 
hydrolytic  cleavage  product  of  the  keratin  substances.  The  last-men- 
tioned investigator  was  also  able  to  detect  thioglycolic  acid  in  the  cleavage 
products  of  wool. 

Bodies  occur  in  the  animal  kingdom  which  form  to  a  certain  extent 
intermediate  substances  between  coagulated  protein  and  keratin.  C.  Th. 
Morner  ^  has  detected  such  a  body  {alhumoid)  in  the  tracheal  cartilage, 
which  forms  a  net-like  trabecular  tissue.  This  substance  appears  to  be 
related  to  the  keratins  on  account  of  its  solubilities  and  the  quantity  of 
the  sulphur  (lead-blackening)  it  contains,  while  according  to  its  solubility 
in  gastric  juice  it  must  stand  close  to  the  proteins.  Another  substance, 
more  similar  to  keratin,  is  the  horny  layer  in  the  gizzard  of  birds.  According 
to  J.  Hedenius  ^  this  substance  is  insoluble  in  gastric  or  pancreatic  juice 
and  acts  quite  like  keratin.  It  contains  only  1  per  cent  sulphur  and  yields 
on  decomposition  only  a  very  little  tyrosine  but  considerable  leucine. 

Keratin  is  amorphous  or  takes  the  form  of  the  tissues  from  which  it  was 
prepared.  On  heating  it  decomposes  and  generates  an  odor  of  burnt  horn. 
It  is  insoluble  in  water,  alcohol,  or  ether.  On  heating  with  water  to  150- 
200°  C.  it  dissolves.  It  also  dissolves  gradually  in  caustic  alkalies,  espe- 
cially on  heating.  It  is  not  dissolved  by  artificial  gastric  juice  or  by  tryp- 
.sin  solutions.  Keratin  gives  the  xanthoproteic  reaction,  as  well  as  the 
reaction  with  Millon's  reagent,  although  the  latter  is  not  always  typical. 


Jenaischen  Gesellsch.  f.  Med.  u.  Naturwissensch.,  1886;    Bauer,  Zeitschr.  f.  physiol. 
Chem.,  .3.}. 

'  Zeitschr.  f.  physiol.  Chem.,  3G,  which  contains  also  the  older  literature. 

2  Morner,  ibid.,  34  and  42;    EmmerHng,  Ref.  in  Chemiker  Zeitung,  1894. 

3  Chem.  Centralbl.  II.,  1905. 

♦Suter,  Zeitschr.  f.  physiol.  Chem.,  20;   Morner,  ibid.,  42;  Friedmann,  Hofmeister's 
Beitrage,  2. 

^  See  Maly's  Jahresber. ,  18. 
*Skand.  Arch.  f.  Physiol.,  S. 


ELASTIN.  75 

In  the  preparation  of  keratin  a  finely  divided  horny  structure  is  treated 
first  with  boihng  water,  then  consecutively  with  diluted  acid,  pepsin- 
hydrochloric  acid,  and  alkaline  trypsin  solution,  and,  lastly,  with  water, 
alcohol,  and  ether. 

Elastin  occurs  in  the  connective  tissue  of  higher  animals,  sometimes  in 
such  large  quantities  that  it  forms  a  special  tissue.  It  occurs  most  abun- 
dantly in  the  cervical  ligament  (ligamentum  nuchse). 

Elastin  used  to  be  generally  considered  as  a  sulphur-free  substance. 
According  to  the  investigations  of  Chittenden  and  Hart,  it  is  a  question 
whether  or  not  elastin  does  not  contain  sulphur,  which  is  removed  by  the 
action  of  the  alkali  in  its  preparation.  H.  Schwarz  has  been  able  to 
prepare  an  elastin  containing  sulphur  from  the  aorta  by  another  method, 
and  this  sulphur  can  be  removed  by  the  action  of  alkalies,  without  changing 
the  properties  of  the  elastin ;  and  recently  Zoja,  Hedin,  Bergh,  and  Richards 
and  GiEs  ^  have  found  that  elastin  contains  sulphur.  The  most  trust- 
worthy analyses  of  elastin  from  the  cer\'ical  ligament  (Nos.  1  and  2)  and 
from  the  aorta  (No.  3)  have  given  the  following  results,  which  compare 
well  with  each  other: 

s  o 

....       21  .94       (HORBACZEWSKI  ^) 

....     21 .  79     (Chittenden  and  Hart) 
0.38     (H.  Schwarz) 

Zoja  found  0.276  per  cent  sulphur  and  16.96  per  cent  nitrogen  in  elastin. 
Hedin  and  Bergh  found  different  quantities  of  nitrogen  in  aorta-elastin, 
depending  upon  whether  Horbaczewski's  or  Schwarz's  method  was 
used  in  its  preparation.  In  the  first  case  they  found  15.44  per  cent  nitro- 
gen and  0.55  per  cent  sulphur,  and  in  the  other  14.67  per  cent  nitrogen 
and  0.66  per  cent  sulphur.  Richards  and  Gies  found  0.14  per  cent  sulphur 
and  16.87  per  cent  nitrogen  in  elastin.  Abundant  leucine,  but  very  little 
tyrosine,  some  glycocoll,  and  perhaps  aminovalerianic  acid,  but  no  aspartic 
acid  or  glutamic  acid,  used  to  be  considered  amongst  the  hydrolytic  cleavage 
products  of  elastin.  Abderhalden  and  Schittenhelm  ^  have  obtained 
glycocoll  25.75;  leucine  21.38;  alanine  6. 58;  phenylalanine  3.89;  a-pro- 
line  1.74;  glutamic  acid  0.76,  and  aminovalerianic  acid  1.0  per  cent.  The 
three  hexone  bases  have  been  obtained,  but  only  in  very  small  amounts, 
so  that  the  basic  nitrogen  represents  only  3.34  per  cent  of  the  total  nitro- 
gen (Richards  and  Gies).  This  fact  and  the  very  low  sulphur  content 
make  it  questionable  whether  the  elastin  is  a  unit  body. 

'  Chittenden  and  Hart,  Zeitschr.  f.  Biologic,  25;  Schwarz,  Zeitschr,  f,  physiol. 
Chem.,  18;  Zoja,  ibuL,  23;  Bergh,  ibid.,  25;  Hedin,  ibid.;  Richards  and  Gies,  Amer. 
Journ.  of  Physiol.,   ". 

^Zeitschr.  f.  physiol.  Chem.,  (5. 

^  Ibid.,  41. 


C 

h 

Is 

r 

1. 

54, 

32 

6.99 

16, 

75 

2. 

54 

,24 

7.27 

16. 

70 

3. 

53 

.96 

7,03 

16, 

,67 

76  THE  PROTEIN  SUBSTANCES. 

On  putrefaction  by  anaerobic  micro-organisms,  Zoja  found  carbon 
dioxide,  hydrogen,  methane,  mercaptan,  butyric  acid,  valerianic  acid, 
ammonia,  and  possibly  also  phenylpropionic  acid  and  aromatic  oxyacids. 
Indol  and  skatol  have  not  been  found  in  putrefaction,^  but  Schwarz,  on 
the  contrary,  obtained  indol,  skatol,  benzene,  and  phenols  on  fusing  aorta- 
elastin  with  caustic  potash.  On  heating  with  water  in  closed  vessels, 
on  boiling  with  dilute  acids,  or  by  the  action  of  proteolytic  enzymes,  the 
elastin  dissolves  and  splits  into  two  chief  products,  called  by  Horbac- 
ZEWSKi  hemielastin  and  elastinpeptone.  According  to  Chittenden  and 
Hart,  these  products  correspond  to  two  proteoses  designated  by  them 
protoelastose  and  deuteroelastose.  The  first  is  soluble  in  cold  water  and 
separates  out  on  heating,  and  its  solution  is  precipitated  by  mineral  acids  as 
well  as  by  acetic  acid  and  potassium  ferrocyanide.  The  aqueous  solution 
of  the  other  does  not  become  cloudy  on  heating,  and  is  not  precipitated  by 
the  above-mentioned  reagents.  According  to  Richards  and  Gies,  elastoses, 
especially  protoelastoses,  and  true  peptone  are  formed,  the  latter  only  to  a 
slight  extent. 

Pure  elastin  when  dry  is  a  yellowish-white  powder;  in  the  moist  state  it 
appears  like  yellowish-white  threads  or  membranes.  It  is  insoluble  in 
water,  alcohol,  or  ether,  and  shows  a  resistance  toward  the  action  of 
chemical  reagents.  It  is  not  dissolved  by  strong  caustic  alkalies  at  the 
ordinary  temperature  and  only  slowly  at  the  boiling  temperature.  It  is 
very  slowly  attacked  by  cold  concentrated  sulphuric  acid,  but  it  is  relatively 
easily  dissolved  on  warming  with  strong  nitric  acid.  Elastins  of  different 
origins  act  differently  with  cold  concentrated  hydrochloric  acid;  for  instance, 
elastin  from  the  aorta  dissolves  readily  therein,  while  elastin  from  the 
ligamentum  nuchse,  at  least  from  old  animals,  dissolves  with  difficulty. 
Elastin  is  more  readily  dissolved  by  warm  concentrated  hydrochloric  acid. 
It  responds  to  the  xanthoproteic  reaction  and  to  that  with  Millon's  reagent. 

On  account  of  its  great  resistance  to  chemical  reagents,  elastin  may  be 
prepared  (best  from  the  ligamentum  nuchse)  in  the  following  way:  First 
boil  with  water,  then  with  1  per  cent  caustic  potash,  then  again  with  water, 
and  lastly  with  acetic  acid.  The  residue  is  treated  with  cold  5  per  cent 
hydrochloric  acid  for  twenty-four  hours,  carefully  washed  with  water, 
boiled  again  with  water,  and  then  treated  with  alcohol  and  ether. 

In  regard  to  the  methods  used  by  Schwarz  and  by  Richards  and  Gies, 
which  are  somewhat  different,  we  refer  to  the  original  publications. 

Collagen,  or  gelatine-forming  substance,  occurs  very  extensively  in 
vertebrates.  The  flesh  of  cephalopods  is  also  said  to  contain  collagen.^ 
Collagen  is  the  chief  constituent  of  the  fibrils  of  the  connective  tissue  and 
(as  ossein)  of  the  organic  substances  of  the  bony  structure.     It  also  occurs 

'See  Walchli,  Journ.  f.  prakt    Chem.  (N.  F.),  1". 
*  Hoppe-Seyler.  Physiol.  Chem.,  p.  97. 


6.47 

17.86 

24 

.92 

(Hofmeister) 

6.80 

17.97 

0. 

t 

25.13 

(Chittenden) 

6.56 

17.81 

0. 

26 

25.26 

(van  N.\me) 

6.71 

17.90 

0, 

57 

24.33 

(Richards  and  GiEs) 

6.76 

17.68 

(Faust) 

COLL AG EX.  77 

in  the  cartilaginous  tissues  as  chief  constituent;  but  it  is  here  mixed  with 
other  substances,  producing  what  was  formerly  called  chondrigen.  Col- 
lagen from  different  tissues  has  not  quite  the  same  composition,  and  prob- 
ably there  are  several  varieties  of  collagen. 

By  continued  boiling  with  water  (more  easily  in  the  presence  of  a 
little  acid)  collagen  is  converted  into  gelatine.  Hofaieister  ^  found  that 
gelatine  on  being  heated  to  130°  C.  is  again  transformed  into  collagen;  and 
this  last  may  be  considered  as  the  anhydride  of  gelatine.  Collagen  and 
gelatine  have  about  the  same  composition.^ 

c  H  N         s  o 

Collagen 50 .  75 

Gelatine  (commercial).  ...  49.38 

Cielatine  from  tendons..  .  .  50.11 

Gelatine  from  ligaments.  .  50.49 

Fish  glue 48 .  69 

Gelatines  of  different  origin  show  a  somewhat  variable  composition, 
which  seems  to  indicate  the  occurrence  of  different  coUagens.  It  is  diffi- 
cult to  say  whether  the  variable  content  of  sulphur  is  due  to  a  contam- 
ination with  a  substance  rich  in  sulphur  or  to  a  splitting  off  of  loosely  com- 
bined sulphur  during  the  purification.  C.  JMorner  ^  has  prepared  a  typical 
gelatine  containing  only  0.2  per  cent  of  sulphur  by  a  method  which  elim- 
inated any  possible  changes  due  to  reagents. 

Sadikoff^  has  prepared  gelatines  by  various  methods  from  tendons 
and  from  cartilage.  Those  from  tendons,  some  of  which  were  prepared 
after  previous  tryptic  digestion,  some  after  treatment  with  0.25  per  cent 
caustic  potash,  and  some  after  treatment  with  sodium  hydroxide  and  then 
carbonate,  showed  somewhat  different  physical  properties  among  each 
other,  but  had  about  the  same  elementary  composition,  with  0.34-0.526  per 
cent  sulphur.  Sadikoff  seems  to  think  that  the  gelatines  prepared  up 
to  this  time  were  perhaps  not  unit  bodies  but  were  possibly  mixtures. 
The  bodies  prepared  by  Sadikoff  from  cartilage  he  calls  gluteins,  because 
they  were  essentially  different  from  the  other  gelatines  or  glutins.  They 
were  poorer  in  carbon  and  nitrogen,  17.7  to  17.87  per  cent,  but  somewhat 
richer  in  sulphur,  0.53-0.712  per  cent,  than  the  tendon  glutin.  The  glu- 
teins differ  also  from  the  glutins  in  that  on  boiling  with  a  mineral  acid 
they  have  a  faint  reducing  action,  and  also  in  that  they  give  a  color  reac- 
tion with  phloroglucin-hydrochloric  acid.  The  glutins  differ  from  the 
gluteins  by  a  different  behavior  with  certain  salts. 

^  Zeitschr.  f.  physiol.  Chem.,  2. 

2  Hofmeister,  1.  c;  Chittenden  and  SoIIey,  Journ.  of  Physiol.,  12;  van  Name, 
Journ.  of  Exper.  Med.,  2;  Richards  and  Gies,  Amer.  Journ.  of  Physiol.,  S;  Faust, 
Arch.  f.  exp.  Path.  u.  Pharm.,  41. 

5  Zeitschr.  f.  physiol.  Chem.,  28. 

*Ibul  ,  39  and  41. 


78  THE  PROTEIN  SUBSTANCES. 

The  decomposition  products  of  the  collagens  are  the  same  as  those  of 
the  gelatines.  Besides  the  leucine,  glycocoll,  aspartic  acid,  and  glutamic 
acid  found  by  the  earlier  investigators  as  hydrolytic  cleavage  products, 
E.  Fischer  and  collaborators  ^  have  obtained  alanine,  phenylalanine,  and 
a-proline.  Gelatme  does  not  give  any  tyrosine,  but  does  ^neld  considerable 
glycocoll  (16.5  per  cent  according  to  E.  Fischer),  which  because  of  its 
.sweetish  taste  has  received  the  name  gelatine-sugar.  Skraup  ^  has  obtained 
on  the  h^'drolytic  cleavage  of  gelatine  a  crystalline  acid  having  the  for- 
mula C12H25N5O10,  which  he  calls  glviinic  acid.  Gelatine  yields  consider- 
able basic  nitrogen,  according  to  Hausmaxx  ^  3o.S3  per  cent  of  the  total  nitro- 
gen. DRECHSELand  Fischer  found  lysine;  Hedix,  Kossel  and  Kutscher  * 
found  also  arginine,  which  amounted  to  9.3  per  cent  (Kossel  and  Kutscher). 
On  putrefaction  gelatine  gives  neither  tyrosine,  indol,  nor  skatol.  According 
to  Seltrexxy  ^  it  yields  phenylpropionic  acid  and  phenylacetic  acid. 
The  aromatic  group  in  gelatine  is  therefore,  as  directly  shown  by  Fischer 
(see  above)  and  also  by  Spiro,^  represented  by  phenylalanine. 

On  the  oxidation  of  gelatine  ^\'ith  potassium  permanganate,  Seemaxx 
obtained,  besides  volatile  fatty  acids  (formic,  acetic,  butyric  acids),  benzoic 
acid,  oxalic  acid,  .succinic  acid,  oxaluramide  and  probably  also  oxaluric 
acid.     Zickcraf  ''  produced  guanidine  from  the  arginine. 

Collagen  is  insoluble  in  water,  salt  solutions,  and  dilute  acids  and  alka- 
lies, but  it  swells  up  in  dilute  acids.  By  continued  boiling  with  water  it  is 
converted  into  gelatine.  It  is  dissolved  by  the  gastric  juice  and  also  by  the 
pancreatic  juice  (trypsin  solution)  when  it  has  previously  been  treated  with 
acid  or  heated  with  water  above  70°  C.^  By  the  action  of  ferrous  .sul- 
phate, corrosive  sublimate,  or  tannic  acid,  collagen  shrinks  greatly.  Col- 
lagen treated  by  these  bodies  does  not  putrefy,  and  tannic  acid  is  there- 
fore of  great  importance  in  the  preparation  of  leather. 

Gelatine  or  glutin  is  colorless,  amorphous,  and  transparent  in  thin  layers. 
It  swells  in  cold  water  without  dissolving.  It  dissolves  iii  warm  water, 
forming  a  sticky  liquid,  which  solidifies  on  cooling  when  sufficiently  con- 
centrated. As  Pauli  and  Roxa  ^  have  .shown,  various  bodies  may  have 
a  different  influence  upon  the  gelatinization-point  of  a  gelatine  solution; 


'  Fischer,  Levene  and  Aders,  Zeitschr.  f.  physiol    Chem.,  35.      In  regard  to  the 
older  researches,  see  O.  Cohnheim,  Chemie  der  Eiweisskorper,  2.  Aufl.,  1904. 
^  Monatshefte  f.  Chem.,  26. 
'  Zeitschr.  f.  physiol.  Chem.,  2". 

*  Drechsel,  Arch.  f.  Anat   u.  Physiol.,  1891;   Hedin,  Zeitschr.  f.  physiol.  Chem.,  21; 
Kossel  and  Kutscher,  ibid.,  31, 

5  Monatshefte  f.  Chem.,  10. 

*  Hofmeister's  Beitrage,  1. 

'  Seemann,  Zeitschr.  f.  physiol.  Chem.,  44;    Zickgraf,  ibid.,  41. 

*  Kiihne  and  Ewald,  Verh.  d.  Naturhist    Med.  Vereins  in  Heidelberg,  1877,  1. 

*  Hofmeister's  Beitraeo.  2. 


GELATINE.  79 

thus  certain  substances  such  as  sulphates,  citrates,  acetates,  and  glycerine 
may  accelerate,  while  the  chlorides,  chlorates,  bromides,  alcohol,  and  urea 
retard  this  power. 

Gelatine  solutions  are  not  precipitated  on  boiling,  nor  by  mineral  acids, 
acetic  acid,  alum,  basic  lead  acetate,  nor  metallic  salts  in  general.  A  gela- 
tine solution  acidified  with  acetic  acid  ma}'  be  precipitated  by  potassium 
farrocj-anide  on  carefully  adding  the  reagent.  Gelatine  solutions  are  precipi- 
tated by  tannic  acid  in  the  presence  of  salt;  by  acetic  acid  and  common 
salt  in  substance;  mercuric  chloride  in  the  presence  of  HCl  and  NaCl; 
metaphosphoric  acid  and  phosphomolybdic  acid  in  the  presence  of  acid;  and 
lastly  also  by  alcohol,  especially  when  neutral  salts  are  present.  Gelatine 
solutions  do  not  diffuse.  Gelatine  gives  the  biuret  reaction,  but  not  Adam- 
KiEwicz's.  It  gives  ^IiLLOx's  reaction  and  the  xanthoproteic  reaction 
so  faintly  that  they  probably  occur  from  impurities  consisting  of  pro- 
teids.  According  to  C.  ^Iorxer,  pure  gelatine  gives  a  beautiful  ^Iillox's 
reaction,  if  not  too  much  reagent  is  added.  In  the  other  case  no  reaction 
or  only  a  faint  one  is  obtained. 

By  continued  boiling  ^^•ith  water  gelatine  is  converted  into  a  non-gelat- 
inizing modification  called  ,5-glutin  by  Nasse.  According  to  Xasse  and 
Kruger  the  specific  rotator}^  power  is  hereby  reduced  from  — 167.5°  to 
about  — 136°.^  On  prolonged  boiling  with  water,  especially  in  the  presence 
of  dilute  acids,  also  in  the  gastric  or  trj-ptic  digestion,  the  gelatine  is  trans- 
formed into  gelatine  proteoses,  so-called  gelatoses  and  gelatine  peptones, 
which  diffuse  more  or  less  readily. 

According  to  Hofmeister  two  new  substances,  semighitin  and  hemi- 
collin,  are  formed.  The  former  is  insoluble  in  alcohol  of  70-80  per  cent 
and  is  precipitated  by  platinum  chloride.  The  latter,  which  is  not  pre- 
cipitated by  platinum  chloride,  is  soluble  in  alcohol.  Chittenden  and 
SoLLEY^  have  obtained  in  the  peptic  and  tryptic  digestion  a  proto-  and 
a  deiiterogelatose,  besides  a  true  peptone.  The  elementary-  composition  of 
these  gelatoses  does  not  essentially  differ  from  that  of  the  gelatine. 

According  to  Levene  the  proto-  as  well  as  the  deuterogelatoses  yield 
a  larger  amount  of  glycocoll,  as  much  as  20.3  per  cent,  than  the  gelatine 
itself.  On  prolonged  tryptic  digestion  a  further  demolition  takes  place,  so 
that  the  peptone  3-ields  only  about  the  same  amount  of  glycocoll  as  the 
gelatine.  Some  leucine  and,  as  it  appears,  also  some  glutamic  acid  and 
phenylalanine  are  split  off.  Quite  a  considerable  splitting  off  of  NH3  also 
takes  place  (Levene  and  Stookey).^     Paal  ^has  prepared  gelatine-peptone 


'  Xasse  and  Kriiger .  Maly's  Jahresber.,  19,  p.  29.  In  regard  to  the  rotation  of  ;9-glutin, 
see  Framni,  Pfliiger's  Arch.,  6S. 

-Hofmeister,  1.  c;    Chittenden  and  SoUey,  1.  c. 

^  Levene,  Zeitsehr.  f.  physioL  Chcm.,  37;   Levene  and  Stookey,  ibid.,  41. 

''  Ber.  d.  devitsch.  chem.  Ge.sellsch.,  2o. 


80  THE  PROTEIN  SUBSTANCES. 

"hydrochlorides  from  gelatine  by  the  action  of  dilute  hydrochloric  acid. 
These  salts  a"8  partly  soluble  in  ethyl  and  methyl  alcohol,  and  partly  insolu- 
ble therein.  The  peptones  obtained  from  these  salts  contain  less  carbon  and 
more  hydrogen  than  the  gelatine  from  which  they  originated,  showing  that 
hydration  has  taken  place.  The  molecular  weight  of  the  gelatine  peptone  as 
determined  by  Paal,  by  Raoult's  cryoscopic  method,  was  200  to  352,  while 
that  for  gelatine  was  878  to  960.  The  gelatine  peptones  isolated  by  Sieg- 
fried and  his  pupils  Scheermesser  ^  and  Kruger,  and  which  have  already 
been  mentioned,  are  of  the  greatest  interest. 

Collagen  (contaminated  with  mucoid)  may  be  obtained  from  bones  by 
extracting  them  with  hydrochloric  acid  (which  dissolves  the  earthy  phos- 
phates) and  then  carefully  washing  the  acid  out  with  water.  It  may  be 
obtained  from  tendons  by  extracting  with  lime-water  or  dilute  alkali 
(which  dissolve  the  proteids  and  mucin)  and  then  thoroughly  washing  with 
\/ater.  Gelatine  is  obtained  by  boiling  collagen  with  water.  The  finest 
<;ommercial  gelatine  always  contains  a  little  proteid,  which  may  be  removed 
by  allowing  the  finely  divided  gelatine  to  swell  up  in  water  and  thoroughly 
extracting  with  large  quantities  of  fresh  water.  Then  dissolve  in  warm 
water  and  precipitate  with  alcohol. 

Collagen  may  also  be  purified  from  proteids,  as  suggested  by  van  Name, 
by  digesting  with  an  alkaline  trypsin  solution  or  by  extracting  the  gelatine 
for  many  days  with  1-5  p.  m.  caustic  potash,  as  suggested  by  C.  Morner. 
The  typical  properties  of  gelatine  are  not  changed  by  this. 

Chondrin  or  cartilage  gelatine  is  only  a  mixture  of  gelatine  with  the  specific 
•constituents  of  the  cartilage  and  their  transformation  products. 

Reticulin.  The  reticular  tissues  of  the  lymphatic  glands  contain  a 
variety  of  fibres  which  have  also  been  found  by  Mall  in  the  spleen,  intestinal 
mucosa,  liver,  kidne3's,  and  lungs.  These  fibres  consist  of  a  special  sub- 
stance, reticulin,  investigated  by  Siegfried.^ 

Reticulin  has  the  following  composition:  C  52.88;  H  6.97;  N  15.63; 
S  1.88;  P  0.34;  ash  2.27  per  cent.  The  phosphorus  occurs  in  organic  com- 
bination. It  yields  no  tyrosine  on  cleavage  with  hydrochloric  acid.  It  yields, 
■on  the  contrary,  sulphuretted  hydrogen,  ammonia,  lysine,  arginine,  and 
aminovalerianic  acid.  On  continued  boiling  with  water,  or  more  readily 
with  dilute  alkalies,  reticulin  is  converted  into  a  body  which  is  precipitated 
by  acetic  acid,  and  at  the  same  time  phosphorus  is  split  off. 

Reticulin  is  insoluble  in  water,  alcohol,  ether,  lime-water,  sodium 
•carbonate,  and  dilute  mineral  acids.  It  is  dissolved,  after  several  weeks, 
on  standing  with  caustic  soda  at  the  ordinary  temperature.  Pepsin-hydro- 
chloric acid  or  trypsin  does  not  dissolve  it.  Reticulin  responds  to  the  biuret, 
xanthoproteic,  and  Adamkiewicz's  reactions,  but  not  to  ^Iillon's  reagent. 


*  Zeitschr.  f.  physiol.  Chem.,  37  and  •41;    Kruger,  1.  c.     See  foot-note  3,  p.  57. 

^  Mall,  Abhandl.  d.  math.-phys.  Klasse  d.  Kgl.  sachs.  Gesellsrh.  d.  Wiss.,  1891; 
Siegfried,  Ueber  die  chem.  Eigensch.  der  retikulirten  Gewebe,  Habil.-Schrift,  Leipzig, 
1892. 


SKELETINS.  81 

According  to  Tebb  reticulin  is  only  a  somewhat  changed,  impure  collagen, 
but  this  is  disputed  by  Siegfried.' 

It  may  be  prepared  as  follows,  according  to  Siegfried:  Digest  intes- 
tinal mucosa  with  trypsin  and  alkali.  Wash  the  residue,  extract  with 
ether,  and  digest  again  with  trypsin  and  then  treat  with  alcohol  and  ether. 
On  careful  boiling  with  water  the  collagen  present  either  as  contamination 
or  as  a  combination  with  reticulin  is  removed.  The  thoroughly  boiled 
residue  consists  of  reticulin. 

Ichthylepidin  is  an  organic  compound,  so  called  by  C.  Morner,^  which  occurs 
■with  collagen  in  fish-scales  and  forms  about  i  of  their  organic  substance.  This 
compound,  with  15.9  per  cent  nitrogen  and  1.1  per  cent  sulphur,  stands  on 
account  of  its  properties  rather  close  to  elastin.  It  is  insoluble  in  cold  and  hot 
water,  as  well  as  in  dilute  acids  and  alkalies  at  the  ordinary  temperature.  On 
boiling  with  these  it  dissolves.  Pepsin-hydrochloric  acid,  as  well  as  an  alkaline 
trypsin  solution,  also  dissolves  it.  It  responds  beautifully  with  Millox's 
reagent,  the  xanthoproteic  reaction,  and  the  biuret  test.  At  least  a  part  of 
the  sulphur  is  split  off  by  the  action  of  alkali. 

As  skeletins,  Krukenberg^  has  designated  a  number  of  nitrogenized 
substances  which  form  the  skeletal  tissue  of  various  classes  of  invertebrates. 
These  substances  are  chitin,  spongin,  conchiolin,  cornein,  and  fibroin  (silk). 
Of  these  chitin  does  not  belong  to  the  protein  substances,  and  fibroin 
(silk)  is  hardly  to  be  classed  as  a  skeletin.  Only  those  so-called  skeletins 
will  he  discussed  that  actually  belong  to  the  protein  group. 

Spongin  forms  the  chief  mass  of  the  ordinary  sponge.  It  gives  no  gelatine.  On 
boiling  with  acids,  according  to  the  older  statements,  it  yields  leucine  and  glyco- 
coll,  but  not  tyrosine.  Zalocostas  claims  to  have  found  tyrosine  and  also  amino- 
isovalerianic  acid  and  glucalanine  (C5Hi2N204).  After  Hundeshagen  had  shown 
the  occurrence  of  iodine  and  bromine  in  organic  combination  in  different  sponges 
and  designated  the  albumoid  containing  iodine,  iodospongin,  Harnack^  later  iso- 
lated from  the  ordinary  sponge,  by  cleavage  with  mineral  acids,  an  iodospongin 
which  contained  about  9  per  cent  iodine  and  4.5  per  cent  sulphur.  On  the  hydrol- 
ysis of  spongin  Abderhalden  and  Strauss  ^  obtained  abundance  of  glutamic  acid, 
18.1,  and  glycocoll,  13.9  per  cent,  also  leucine,  7.5,  a-proline,6.3,and  aspartic  acid, 
4.1  per  cent.  Very  remarkable  was  the  fact  that  neither  tyrosine  nor  phenylalanine 
could  be  detected.  Strauss  *  has  obtained  sponginoses  of  various  kinds  from 
spongin  by  dilute  acids.  The  heterosponginose  contained  the  greater  part  of 
the  iodine  and  sulphur,  while  the  deuterosponginose  contained  the  carbohydrate 
groups.  Iodospongin  is  considered  as  a  derivative  of  the  heterosponginose. 
Conchiolin  is  found  in  the  shells  of  mussels  and  snails  and  also  in  the  egg-shells  of 
these  animals.  It  yields,  according  to  Wetzel,^  glycocoll,  leucine,  and  abundance 
of  tyrosine.  The  quantity  of  diamino-nitrogen  amounts  to  8.7  per  cent  and  the 
amide  nitrogen  3.47  per  cent  (from  the  shell  of  pinna).     The  Byssus  contains  a 

»Tebb,  Journ.  of  Physiol.,  27;    Siegfried,  ibid.,  28. 

'  Zeitschr.  f.  physiol.  Chem.,  24  and  37.     See  also  Green  and  Tower,  ibid.,  35. 
'  Grundziige  einer  vergl.  Physiol,  d.  thier.  Geriistsubst.,  Heidelberg,  1885. 
*  Zalocostas,  Compt.  rend.,  107;    Hundeshagen,  Maly's  Jahresber.,  25;    Harnack, 
Zeitschr.  f.  physiol.  Chem.,  24. 
'  Zeitschr.  f.  physiol.  Chem.,  48. 
'  Biochem.  Centralbl.,  3. 
'Zeitschr,  f.  physiol.  Chem.,  29,  and  Centralbl.  f.  Physiol,,  13,  113. 


82 


THE  PROTEIN  SUBSTANCES. 


substance,  closely  relat3d  to  conchiolin,  which  is  soluble  with  difficulty.  Comein 
forms  the  axial  system  of  the  Antipathes  and  Gorgonia.  It  gives  leucine  and  a 
crystallizable  substance,  cornicrystalUne.  According  to  Drechsel  the  axial  sys- 
tem of  the  Gorgonia  cavolini  contains  nearly  8  per  cent  of  the  dry  substance  as 
iodine.  The  iodine  occurs  in  organic  combination  with  an  iodized  albumoid,  gor- 
gonin,  which  is  a  comein.  Drechsel  obtained  leucine,  tyrosine,  lysine,  ammonia, 
and  an  iodized  amino-acid,  iodogorgonic  acid,  as  cleavage  products  of  gorgonin. 
According  to  Wheeler  and  Jamieson  '  iodogorgonic  acid  is  diiodotyrosine,  prob- 
ably 3,5-diiodotyrosine,  C6H2(CH,CH(NH2)COOH)(OH)I.,  and  was  prepared  by 
them  by  the  action  of  iodine  upon  tyrosine  and  alkali.  Henze  '  could  obtain  this 
acid  only  in  very  small  quantities,  and  by  acid  cleavage  of  gorgonin  he  obtained 
the  three  hexone  bases,  abundance  of  tyrosine,  and  very  little  1  sucine.  On  cleavage 
with  barium  hydrate  he  obtained  only  lysine  besides  tyro.iine  and  glycocoU  in 
larger  amounts. 

Fibroin  and  sericin  are  the  two  chief  constituents  of  raw  silk.  By  the  action 
of  boiling  water  the  sericin  (silk  gelatine)  dissolves  and  can  be  obtained  by  a 
method  suggested  by  Bondi,^  while  the  more  difficultly  soluble  fibroin  remains 
undissolved  in  the  shape  of  the  original  fibre.  The  sericin,  whose  sufficiently 
concentrated  hot  solution  gelatinizes  on  cooling,  is  precipitated  by  mineral  acids, 
several  metallic  salts,  and  by  acetic  acid  and  potassium  ferrocyanide.  As  cleavage 
products  E.  Fischer  and  Skita  obtained  alanine,  serine,  very  little  glycocoll, 
tyrosine,  arginine,  and  probably  also  lysine.  Leucine  had  been  found  previously. 
From  fibroin  they  obtained,  besides  the  previously  known  cleavage  products, 
glycocoll,  tyrosine,  and  alanine  (Weyl  *),  also  leucine,  phenylalanine,  serine, 
a-proline  (Fischer),  and  a  small  amount  of  arginine.  The  chief  products  were 
glycocoll,  36  per  cent,  alanine,  21  per  cent,  and  tyrosine,  10  per  cent.  The  com- 
position of  the  above-mentioned  albuminoids  is  as  follows :  ^ 


Gjnchiolin  (from  the  shells  of  pinna).  .  .    52.70 

' '  (from  snail  eggs) 50 .  92 

Spongin 46 .  50 

"      48.75 

Cornein 48.96 

Fibroin 48.23 

48.30 

Sericin 44.32 

44.50 


6.54 

16.60 

0 

85 

(Wetzel) 

6.88 

17.86 

0 

31 

(Krukenberg) 

6.30 

16.20 

0 

50 

(Croockewitt) 

6.35 

16.40 

(Posselt) 

5.90 

16.81 

(Krukenberg) 

6.27 

18.31 

(Cramer) 

6.50 

19.20 

(Vignon) 

6.18 

18.30 

(Cramer) 

6.32 

17.14 

(Bondi) 

'  Amer.  Chem.  Journ.,  33. 

^  Drechsel,  Zeitschr.  f.  Biologie,  33;    Henze,  Zeitschr.  f.  physiol.  Chem.,  38. 

^  Zeitschr.  f.  physiol.  Chem.,  34. 

*  Fischer  and  Skita,  ibid.,  33;  Fischer,  ibid.,  39;  Weyl,  Ber.  d.  d.  chem.  Gesellsch.,  21. 

'Krukenberg,  Ber.  d.  d.  chem.  Gesellsch.,  17  and  18,  and  Zeitschr.  f.  Biologie,  22; 
Croockewitt,  Annal.  d.  Chem.  u.  Pharm.,  48;  Posselt,  ibid.,  45;  Cramer,  Journ.  f. 
prakt.  Chem.,  96;   Vignon,  Compt.  rend.,  llo;  Wetzel,  1.  c,  and  Bondi,  1.  c. 


GLYCOCOLL.  83 

Appendix  to  Chapter  II. 

HYDROLYTIC  CLEAVAGE  PRODUCTS  OF  THE  PROTEIN  SUBSTANCES. 
I.    Monamino-acids. 

GlycocoU  (aminoacetic  acid),  C'2H5N02=       ?^L    ^  ,  also  called  glvcine 

or  gelatine  sugar,  is  found  in  the  muscles  of  the  invertebrates,  but  has 
chief  interest  as  a  hydrolytic  decomposition  product  of  protein  bodies, 
especially  gelatine,  fibroin,  and  spongin,  as  well  as  of  hippuric  acid  a<nd 
glycocholic  acid.  It  is  also  formed  in  the  decomposition  of  uric  acid, 
xanthine,  guanine,  and  adenine. 

GlycocoU  has  been  most  abundantly  obtained  thus  far  from  the  protein 
substances  fibroin  ^  (36  per  cent),  elastin^  (25.75  per  cent),  gelatine  and 
gelatoses^  (16.5  and  20.3  per  cent  respectively). 

GlycocoU  forms  colorless,  often  large,  hard  rhombic  crystals  or  four- 
sided  prisms.  The  crystals  have  a  sweet  taste  and  dissolve  readily  in 
cold  water  (4.3  parts).  It  is  insoluble  in  alcohol  and  ether  and  dissolves 
with  difficulty  in  warm  alcohol.  GlycocoU  combines  with  acids  and  alkalies. 
With  the  latter  compounds  we  must  mention  those  with  copper  and 
silver.  GlycocoU  dissolves  cupric  hydrate  in  alkaline  liquids  but  does 
not  reduce  at  boiling  heat.  A  boiling-hot  solution  of  glycocoll  dissolves 
freshly  precipitated  cupric  hydrate,  forming  a  blue  solution,  which,  in 
proper  concentration,  deposits  blue  needles  of  copper  glycocoll  on  cooling. 
The  compound  with  hydrochloric  acid  is  readily  soluble  in  water  but  less 
soluble  in  alcohol. 

SoRENSEN^  finds  that  phosphotungstic  acid  does  not  precipitate  glyco- 
coll from  dilute  solutions  but  only  from  concentrated  ones.  By  the  action  of 
gaseous  HCl  upon  glycocoll  in  absolute  alcohol,  beautiful  crystals  are 
obtained  of  the  hydrochloride  of  glycocoll  ethyl  ester,  which  melts  at  144°  C. 
and  from  which  the  glycocoll  ethyl  ester  can  be  obtained  by  the  method 
suggested  by  E.  Fischer  ^  for  the  separation  of  glycocoll  from  the  other 
amino-acids.  On  shaking  with  benzoyl  chloride  and  caustic  soda,  hippuric 
acid  is  formed,  and  this  is  also  made  use  of  in  different  ways  in  detecting 
and  isolating  glycocoll  (Ch.  Fischer,  Gonnermann,  Spiro  ^).     The  melting- 

'  Fischer  and  Skita,  Zeitschr.  f.  physiol.  Chem.,  33. 
^  Abderhalden  and  Schittenhelm,  ibid.,  41. 

^  Fischer,  Levene  and  Aders,  ibid.,  35;    Levene,  ibid.,  37  and  41. 
*  Meddelelser,  fraa  Carlsberg-laboratoriet,  6,  190.5. 
^  Ber.  d.  d.  chem.  Gesellsch.,  34. 

'  Ch.  Fischer,  Zeitschr.  f.  physiol.  Chem.,  19;  Spiro,  ibid.,  2S;  Gonnermann, 
Pfliiger's  Arch.,  59. 


84  THE  PROTEIN  SUBSTANCES. 

point  of  glycocoll-/3-naphthalenesulphonate  is  156°  (corr.  159°),  of  glycocoU 
4-nitrotoluene-2-sulphonate  177.5°  (corr.  178°),  of  the  phenylisocyanate 
compound  195°,  and  of  the  a-naphthylisocyanate  compound  190.5-191.5°. 

GlycocoU  can  be  best  prepared  from  hippuric  acid  by  boiUng  it  with 
4  parts  dihite  sulphuric  acid  (1:6)  for  ten  to  twelve  hours.  After  cooling 
the  benzoic  acid  is  removed,  the  filtrate  concentrated,  the  remaining  benzoic 
acid  removed  by  extracting  with  ether,  the  sulphuric  acid  precijHtated  by 
BaCOa,  and  the  filtrate  evaporated  to  the  point  of  crystallization.  (In  regard 
to  its  preparation  from  protein  substances  see  below.) 

CH3 
Alanine  (a-aminopropionic  acid),  C3H7N02  =  CH(NH2),  was  first  obtained 

COOH 
by  Weyl  as  a  cleavage  product  of  fibroin.  This  d-alanine  has  been 
isolated  by  E.  Fischer  and  his  collaborators^  still  more  abundantly  from 
fibroin  (21  per  cent)  and  also  from  sericin  (5  per  cent),  horn  substance 
(1.20  per  cent),  gelatine  (0.8  per  cent),  haemoglobin  (2.87  per  cent),  and 
elastin  ^  (6.58  per  cent). 

Alanine  has  a  sweet  taste,  is  readily  soluble  in  water,  and  dissolves  cupric 
hydrate  on  boiling,  producing  copper  alanine,  which  has  a  deep  blue  color. 
The  specific  rotation  of  the  hydrochloride  (9-10  per  cent  solution)  is  (a)D  = 
+  10.3°.  In  regard  to  the  synthetical  preparation  of  {-alanine,  its  separa- 
tion as  the  benzoyl  compound,  and  the  preparation  of  r-alanine  ethyl 
ester  we  must  refer  to  E.  Fischer.^ 

The  d-alanine-/?-naphthalenesulphonate  melts  at  78-80°  (79-81°  corr.), 
the  racemic  alanine-4-nitrotoluene-2-sulphonate  at  96°  (uncorr.),  the 
phenylisocyanate  compound  at  168°,  and  the  a-naphthylisocyanate  com- 
pound at  198°  C. 

CH3CH3 


Aminovalerianic  acid,  C5HiiN02=     •  has  been  detected  several  times 

CH(NH2), 

COOH 
among  the  cleavage  products  of  protein  substances.  Kossel  and  Dakin  ^  ob- 
tained 4.3  per  cent  from  salmine.  The  acid  isolated  by  E.  Fischer  from  horn 
substance  (5.70  per  cent)  and  casein,  as  well  as  that  obtained  by  Schulze  and 
WiNTERSTEiN  ^  from  lupin  sprouts,  seems  to  be  dextrorotatory  a-aminovalerianic 
acid.  The  copper  salt  of  aminovalerianic  acid  is,  according  to  Schulze  and 
WiNTERSTEiN,"  readily  soluble  in  methyl  alcohol. 


^Weyl,  Ber.  d.  d.  chem.  Gesellsch.,  21;  Fischer  and  Skita,  Zeitschr.  f.  physiol. 
Chem.,  33;  Fischer  and  Dorpinghaus,  ibid.,  36;  Fischer,  Levene  and  Aders,  ibid., 
35;    Fischer  and  Abderhalden,  ibid.,  36. 

^  Abderhalden  and  Schittenhelm,  Zeitschr.  f.  physiol.  Chem.,  41. 

3  Ber.  d.  d.  chem.  Gesellsch.,  32  and  34. 

^Zeitschr,  f.  physiol.  Chem.,  41. 

'  Fischer,  iind.,  36  and  33;  Schulze  and  Winterstein,  ibid.,  35. 

""Ibid.A^. 


LEUCINE.  85 

Leucine  (aminocaproic  acid,  or,  more  correctly,  a-aminoisobutylacetic 
CH3CH3 


CH 

acid),C6Hi3N02=    CH2  »   is   produced   from  protein   substances  in 

CH(NH2) 

COOH 
their  hydrolytic  cleavage  by  proteolytic  enzymes,  by  boiling  with  dilute 
acids  or  alkalies  or  by  fusing  with  alkali  hydrates,  and  by  putrefaction. 
Because  of  the  ease  with  which  leucine  (and  tyrosine)  are  formed  in  the 
decomposition  of  protein  substances,  it  is  difficult  to  decide  positively 
whether  these  bodies  when  found  in  the  tissues  are  constituents  of  the  living 
body  or  are  to  be  considered  only  as  decomposition  products  formed  after 
death.  Leucine,  it  seems,  has  been  found  as  a  normal  constituent  of  the 
pancreas  and  its  secretion,  in  the  spleen,  thymus,  and  lymph  glands,  in  the 
thyroid  gland,  in  the  salivary  glands,  in  the  kidneys  and  in  the  liver.  It  also 
occurs  in  the  wool  of  sheep,  in  dirt  from  the  skin  (inactive  epidermis),  and 
between  the  toes,  and  its  decomposition  products  have  the  disagreeable  odor 
of  the  perspiration  of  the  feet.  It  is  found  pathologically  in  atheromatous 
cysts,  ichthyosis  scales,  pus,  blood,  liver,  and  urine  (in  diseases  of  the 
liver  and  in  phosphorus  poisoning).  Leucine  occurs  often  in  invertebrates 
and  also  in  the  plant  kingdom.  On  hydrolytic  cleavage  various  protein 
substances  yield  different  amounts  of  leucine.  Erlenmeyer  and  Schoffer 
obtained  36-45  per  cent  of  leucine  from  the  cervical  ligament,  Abderiialden 
and  ScHiTTENHELM  2L3S  per  cent  from  elastin,  Cohn  32  per  cent  from 
casein,  and  Nencki  L5-2  per  cent  from  gelatine.  E.  Fischer  and  Abder- 
HALDEN  obtained  20  per  cent  of  leucine  from  haemoglobin,  Fischer  and 
DoRPiNGHAUS  18.3  per  cent  from  horn  substance,  Nencki  1..5-2  per  cent 
from  gelatine,  and  Fischer  and  Skita  1.5  per  cent  from  fibroin.^ 

Leucine  occurs,  like  other  monamino-acids,  in  the  /-,  d-,  and  ^-modifica- 
tions.  The  leucine  obtained  by  cleavage  of  protein  substances  is  generally 
levorotatory  in  watery  solution  and  dextrorotatory  Z-Ieucine  in  acid  solution. 
The  leucine  prepared  synthetically  by  HtJFNER^  from  isovaleraldeh3'de, 
ammonia,  and  hydrocyanic  acid  is  optically  inactive.  Inactive  leucine  may 
also  be  prepared,  as  shown  by  E.  Schulze  and  Bosshard,^  by  the  cleavage 
of  proteins  with  baryta  at  160-180°  C,  or  by  heating  ordinary  leucine  with 
baryta-water  to  the  same  temperature.      The  levorotatory  modification 

'Erlenmeyer  and  Schoffer,  cited  from  Maly,  Chem.  d,  Verdauungssafte,  in  Her- 
mann's Handb.  d.  Physiol.,  5,  Theil  2,  p.  209;  Abderhalden  and  Schittenhehn, 
Zeitschr.  f,  physiol.  Chem.,  41;  Cohn,  ibid.,  22;  Nencki,  Journ.  f.  prakt.  Chem.  (N. 
F.),  15;  Fischer  and  his  collaborators,  see  p.  84,  foot-note  1. 

^  Journ.  f.  prakt.  Chem.  (N.  F.),  1. 

^  See  Zeitschr.  f .  physiol.  Chem.,  9  and  10. 


86  THE  PROTEIN  SUBSTANCES. 

may  be  formed  from  the  inactive  leucine  by  the  action  oi  Penicillum  glauciim. 
On  benzoylating  i-leucine  we  obtain  i-benzoyl  leu  cine,  from  whose  cinchonine 
and  quinidine  salts  first  d-  and  then  Z-benzoy  lieu  cine  are  prepared,  and 
then  by  hydrolytic  cleavage  d-  and  Z-leucine  may  be  obtained  (E.  Fischer). 
On  oxidation  the  leucines  yield  the  corresponding  oxyacids  (leucinic  acids). 
Leucine  is  decomposed  on  heating,  evohang  carbon  dioxide,  ammonia,  and 
amylamine.  On  heating  with  alkalies,  as  also  in  putrefaction,  it  3'ields 
valerianic  acid  and  ammonia. 

Leucine  crystallizes  when  pure  in  shining,  white,  very  thin  plates,  usually 
forming  round  knobs  or  balls,  either  appearing  like  hyaline,  or  with  alter- 
nating light  and  dark  concentric  layers  which  consist  of  radial  groups  of 
crystals.  By  slow  heating,  leucine  melts  and  sublimes  in  white,  woolly 
flakes,  which  are  similar  to  sublimed  zinc  oxide.  At  the  same  time  an  odor 
of  amylamine  is  developed.  Quickly  heated  in  a  closed  capillary  tube,  it 
melts  with  decomposition  at  293-295°. 

Leucine,  as  obtained  from  animal  fluids  and  tissues,  is  verv^  easily  soluble 
in  water  and  rather  easily  in  alcohol.  Pure  leucine  is  soluble  with  difficulty. 
Pure  I-  and  d-leucine  dissolve  in  40-46  parts  water,  more  readily  in  hot 
alcohol,  but  with  difficulty  in  cold  alcohol.  The  t-leucine  is  much  less  solu- 
ble. According  to  Habermann  and  Ehrenfeld  ^  100  parts  of  boiling 
glacial  acetic  acid  dissolve  29.23  parts  of  leucine.  The  specific  rotation  of 
the  ordinary  leucine,  dissolved  in  hydrochloric  acid,  is  about  (a)D=  +17.5°. 

The  solution  of  leucine  in  water  is  not,  as  a  rule,  precipitated  by  metallic 
salts.  The  boiling-hot  solution  may,  however,  be  precipitated  by  a  boiling- 
hot  solution  of  copper  acetate,  and  this  fact  is  made  use  of  in  separating 
leucine  from  other  substances.  If  the  solution  of  leucine  is  boiled  with 
sugar  of  lead  and  then  ammonia  be  added  to  the  cooled  solution,  shining 
crystalline  leaves  of  leucine-lead  oxide  separate.  Leucine  dissolves  cupric 
hydrate,  but  does  not  reduce  on  boiling. 

Leucine  is  readily  soluble  in  alkalies  and  acids.  It  gives  crystalline  com- 
pounds with  mineral  acids.  If  leucine  hydrochloride  is  boiled  with  alcohol 
containing  3-4  per  cent  HCl,  long  narrow  crystalline  prisms  of  leucine  ethyl 
ester  hydrochloride,  melting  at  134°  C,  are  formed  (Rohmann).  The 
same  is  produced  by  the  action  of  gaseous  HCl  upon  leucine  in  alcohol, 
and  the  free  ethyl  ester  can  be  obtained  from  this  by  the  method  suggested 
by  E.  F1SCHER.2  This  ester  can  be  separated  from  the  other  amino-acid 
esters  by  distillation.  The  pure  leucine  can  be  prepared  from  the  ester  by 
boiling  with  water  for  a  long  time.  The  picrate  of  the  leucine  ester  melts  at 
128°  C.  The  phenylisocyanate  compound  of  i-leucine  melts  at  165°  C. 
and  its  anhydride  at  125°  C.  The  a-naphthylisocyanate  compound  melts  at 
163.5°  and  Z-leucine  /3-naphthalenesulphonate  melts  at  67°  (corr.  68°). 


'  Zeitschr.  f.  physiol.  Cheni.,  37. 

^Rohmann,  Ber.  d.  d.  chem.  Gesellsch.,  30;   E.  Fischer,  ibid.,  34. 


ISOLEUCINE    AND    ASPARTIC    ACID.  87 

Leucine  is  recognized  by  the  appearance  of  balls  or  knobs  under  the 
microscope,  by  its  action  when  heated  (sublimation  test),  and  by  its  com- 
pounds, especially  the  hydrochloride  and  picrate  of  the  ethyl  ester  and  the 
phenylisocyanate  compound  of  the  racemic  leucine  obtained  by  heatin;;; 
with  baryta-water,  the  a-naphthylisocyanate  compound  and  the  leucine 
^-naphthalenesulphonate.  Leucine  must  first  be  isolated  before  it  can  be 
detected,  and  this  is  best  done  by  preparing  the  ethyl  ester  and  then  dis- 
tilling it. 

Leucinimide,  CoHzzNaOj  =    ^    "Viz-i  attlt  V^u  n  tt  >  was  first  obtained  by  Ritt- 

LU .  J\  rl  .Lrl .  U4ri9 

HAtJSEN'  in  the  hydrolytic  cleavage  products  on  boiling  proteins  with  acids,  and 

subsequently  by  R.  Corn.     Salaskin  '  obtained  it  in  the  peptic  and  tryptic 

digestion  of  haemoglobin.     As  an   anhydride  of  leucine    (2.5-diacipiperaznie)    it 

is  probably  formed  by  a  secondary  change,  from  leucine. 

It  crystallizes  in  long  needles  and  sublimes  readily.     The  melting-point  has 

not  been  found  constant  in  the  different  cases.     The  leucinimide  (3.6-diisobutyl- 

2.5-diacipiperazine)  prepared  synthetically  by  E.  Fischer  ^  from  leucine  ethyl 

ester  melted  at  271°  C. 

Isoleucine,  an  isomer  of  leucine,  has  recently  been  discovered  by  F.  Ehr- 
LiCH,  but  its  constitution  is  still  unknown.  Ehrlich  first  isolated  it  from 
the  mother-liquor  after  removing  the  sugar  from  molasses,  and  found  it  also 
on  the  hydrolysis  of  several  proteins,  and  considers  it  as  regularly  asso- 
ciated with  ordinary  leucine.  Winterstein  and  Pantanelli  obtained 
it  on  the  hydrolysis  of  the  protein  of  lupin  seeds,  and  it  has  also  been  found 
by  Schulze  and  Winterstein  ^  in  sprouts. 

Isoleucine  is  more  soluble  in  water  than  Z-leucine  (1 :  25.8).  It  is  dextro- 
rotatory in  aqueous  as  well  as  in  acid  solutions  and  in  the  presence  of  hydro- 
chloric acid  it  acts  more  than  twice  as  strongly  as  ordinary  leucine.  In 
aqueous  solution  the  specific  rotation  is  (a:)D=  +9.74°,  in  hydrochloric-acid 
solution  =  +36.8°.  Isoleucine  melts  at  280°,  and  the  benzoyl  compound 
has  a  melting-point  of  116-117°.  Its  copper  salt  is  rather  soluble  in  water 
and,  like  the  copper  salt  of  aminovalerianic  acid,  is  readily  soluble  in  methyl 

alcohol. 

COOH 

Aspartic   Acid   (aminosuccinic  acid),   C4H7N04=^^^  ,  has   been 

COOH 

obtained  on  the  cleavage  of  protein  substances  by  proteolytic  enzymes 
as  well  as  by  boiling  them  with  dilute  mineral  acids.  Hlasiwetz  and 
Habermann  obtained  23.8  per  cent  from  ovalbumin  and  9.3  per  cent 
from  casein,  although  the  product  was  not  quite  pure.     E.  Fischer  and 

'  Ritthausen,  Die  Eiweisskorper  der  Getreidearten,  etc.,  Bonn,  1872;  R.  Cohn, 
Zeitschr.  f.  physiol.  Chem.,  22  and  29;    Salaskin,  ibid.,  32. 

^  Ber,  d    d.  chem.  Gesellsch.,  34. 

^  Felix  Ehrlich  il>id,,  37;  Winterstein  and  Pantanelli,  Zeitschr.  f.  physiol.  Chem., 
45;    Schulze  and  Winterstein   ibid.,  45 


88  THE  PROTEIN  SUBSTANCES. 

his  co-workers  ^  obtained  3.29  per  cent  aspartic  acid  from  haemoglobin, 
2.50  per  cent  from  horn  substance,  and  0.56  per  cent  from  gelatine.  This 
acid  also  occurs  in  secretions  of  sea-snails  (Hexze  ~)  and  is  verj-  widely 
diffused  in  the  vegetable  kingdom  as  the  amide  Asparagixe  (aminosuccinic- 
acid  amide),  which  seems  to  be  of  the  greatest  importance  in  the  develop- 
ment and  formation  of  the  proteins  in  the  plants. 

Aspartic  acid  dissolves  in  256  parts  water  at  10°  C.  and  in  IS. 6  parts 
boiling  water,  and  crystallizes  on  cooling  as  rhombic  prisms.  The  acid' 
prepared  from  protein  substances  is  optically  active,  and  its  4  per  cent  solu- 
tion acidified  with  HCl  has  the  rotation  (q:)d=  +25.7°;  but  it  is  either 
dextrogyrate  or  levogyrate  in  a  watery  solution,  depending  upon  the  tem- 
perature. It  forms  with  copper  oxide  a  crA'stalline  compound  which  is 
soluble  in  boiling-hot  water  and  nearly  insoluble  in  cold  water,  and  which 
may  be  used  in  the  preparation  of  the  pure  acid  from  a  mixture  with  other 
bodies. 

In  regard  to  the  benzoylaspartic  acids  and  the  diethylester  we  must 

refer  to  the  work  of  E.  Fischer  and  his  collaborators.     For  identification 

we  make  use  of  the  analysis  of  the  free  acid  and  the  copper  salts,  as  well 

as  the  specific  rotation. 

COOH 

CH(XH2) 
Glutamic  acid  fa-aminoglutaric  acid),  C5H9N04  =  CH2         ,  is  obtained 

CH2 
COOH 
from  the  protein  sul)stances  under  the  same  conditions  as  the  other  mon- 
amino-acids  and  from  the  peptones  (Siegfried).  Hlasiwetz  and  Haber- 
MANX  obtained  29  i:)er  cent  from  casein  by  cleavage  with  hydrochloric  acid, 
while  KuTSCHER  could  oljtain  only  1.8  per  cent  glutamic  acid  by  cleavage 
with  sulphuric  acid.  Horbaczewski  has  obtained  15-18  per  cent  glu- 
tamic acid  from  gelatine  and  about  the  same  amount  from  horn,  while 
Fischer  and  Dorpixghaus  obtained  only  3  per  cent  from  horn.  Fischer 
and  Abderhaldex  obtained  1.06  per  cent  from  haemoglobin,  Kutscher 
3.66  per  cent  from  thymus  hist  one,  and  Abderhaldex  and  Pregl  ^  ob- 
tained 8  per  cent  from  ovalbumin. 

Glutamic  acid  crystallizes  in  rhomljic  tetrahedra  or  octahedra  or  in 
small  leaves.  It  melts  at  202-203°  C.  with  partial  decomposition.  It 
dissolves  in  100  parts  water  at  16°  C.  and  in  1500  parts  80  per  cent  alcohol. 

'  Hlasiwetz  and  Habermann,  Annal.  d.  Chem.  u.  Pharm.,  159  and  169;  E.  Fischer 
and  collaloorators,  see  foot-note  1,  p.  84. 

^  Ber.  d.  d.  chem.  Gesellsch.,  34. 

^  Hlashvetz  and  Habermann,  1.  c,  159;  Kutscher,  Zeitschr.  f.  physiol.  Chem.,  28 
and  38;  Horbaczewski,  Maly's  Jahre.'-ber.,  10;  Fischer  and  collaborators,  L  c; 
Abderhalden  and  Prcgl,  Zeitschr.  f.  physiol.  Chem.,  46. 


TYROSINE.  89 

It  is  insoluble  in  alcohol  and  ether.  The  rf-glutamic  acid  obtained  from 
proteins  by  boiling  with  an  acid  or  from  the  mother-liquor  from  molasses 
is  dextrorotatory,  and  in  water  has  a  rotation  of  (a)D=  +12.04°  according 
to  AxDRLiK.i  Strong  acids  increase  the  rotation,  and  a  5  per  cent  solution 
of  glutamic  acid  containing  9  per  cent  HCl  has  a  rotation  (a)D=  +31.7°, 
while  that  obtained  by  heating  with  barium  hydrate  is  optically  inactive. 
The  (/-glutamic  acid  forms  a  beautifully  crystalline  combination  with  hydro- 
chloric acid,  which  is  nearly  insoluble  in  concentrated  hydrochloric  acid. 
This  compound  is  used  in  the  isolation  of  glutamic  acid.  On  boiling  with 
cupric  hydrate  a  beautiful  crystalline  copper  salt,  which  is  soluble  with 
difficulty,  is  obtained.  Like  the  monamino-acids  in  general,  glutamic  acid 
is  not  precipitated  by  phosphotungstic  acid.  In  regard  to  the  benzoylglu- 
tamic  acids  and  the  diethylester  we  must  refer  to  the  works  of  Fischer.^ 
The  hydrochloride,  the  a-naphthylisocyanate  of  glutamic  acid  which  melts 
at  236-237°  C.  the  analysis  of  the  free  acid,  and  the  specific  rotation  are 
used  in  its  detection. 

C6H4(OH) 

...  -^      CH., 

Tyrosine  (7>oxypheny.-Q:-ammopropionic  acid).  C9H1 1X03  =  ptt-.^tt  n,  is 

COOH 

produced  from  most  protein  substances  (not  from  gelatine  and  reticulin) 
under  the  same  conditions  as  leucine,  which  it  habitually  accompanies.  The 
largest  quantity  of  tyrosine  obtained  from  animal  proteins  was  obtained 
by  Fischer  and  Skita  from  fibroin,  namely,  10  per  cent.  The  maxi- 
mum obtained  from  thymus  histone  (Kutscher)  was  6.3  per  cent,  from 
horn  substance  (R.  Cohx)  4.6  per  cent,  from  casein  (Reach)  4.55  per  cent, 
from  fibrin  (Kuhxe)  3.86  per  cent,  from  ovalbumin,  seralbumin,  and  ser- 
globulin  (K.  ^Iorxer)  2.4.  2.0,  and  3.0  per  cent  respectively,  from  syntonin 
(Reach)  1.37  per  cent,  from  haemoglobin  (Fischer  and  Abderhaldex) 
1.5  per  cent,  and  from  elastin  (Schwarz^)  0.34  per  cent.  It  is  especially 
found  with  leucine  in  large  quantities  in  old  cheese  (Tvpoi).  form  which 
it  derives  its  name.  Tyrosine  has  not  with  certainty  been  fomid  in  per- 
fectly fresh  organs.  It  occurs  in  the  intestine  in  the  digestion  of  protein 
substances,  and  it  has  about  the  same  physiological  and  pathological  im- 
portance as  leucine. 

Tyrosine  was  prepared  by  Erlexmeyer  and  Lipp  from  p-aminophenyl- 
alanine  bv  the  action  of  nitrous  acid,  and  according  to  another  method  bv 


'  See  Biochem.  Centralbl.,  3,  p.  469. 

M.  c. 

^  Fischer  and  Skita,  1.  c;  Kutscher,  Zeitschr.  f.  physiol.  Chem.,  3S;  R.  Cohn,  ibid., 
26;  Reach,  Virchow's  Arch.,  loN;  Kiihne,  ibid.,  39;  K.  Morner,  Zeitschr.  f.  physiol. 
Chem.,  34;    Fischer  and  Alxlerhalden,  ibid.,  1.  c.;    Schwarz,  ibid.,  18. 


90  THE  PROTEIN  SUBSTANCES. 

Erlenmeyer  and  Halsey.^  On  fusing  with  caustic  alkali  it  fields  p-oxy- 
benzoic  acid,  acetic  acid,  and  ammonia.  On  putrefaction  it  may  yield 
jD-hydrocoumaric  acid,  oxyphenylacetic  acid,  and  p-cresol. 

Naturally  occurring  tyrosine  and  that  obtained  by  the  cleavage  of  pro- 
tein substances  is  generally  Z-tyrosine,  Avhile  that  obtained  by  decomposition 
"vvith  baryta-water  or  prepared  synthetically  is  inactive,  v.  Lippmann^ 
has  obtained  d-tyrosine  from  beet-sprouts.  The  statements  as  to  specific 
rotation  of  tyrosine  are  somewhat  variable.  For  tyrosine  from  jjroteins  E. 
Fischer  has  found  a  rotation  of  (a)D  =  —  12.56  to  13.2°  for  the  hydrochloric- 
acid  solution,  while  Schulze  and  Winterstein^  obtained  higher  results 
using  tyrosine  from  plants,  namely,  (a:)D=  — 16.2°.  These  investigators 
believe  that  when  lower  results  are  obtained  a  contamination  with  racemic 
tyrosine  is  the  cause. 

Tyrosine  in  a  very  impure  state  may  be  in  the  form  of  balls  similar  to 
leucine.  The  purified  tyrosine,  on  the  contrary,  appears  as  colorless,  silky, 
fine  needles  which  are  often  grouped  into  tufts  or  balls.  It  is  soluble  with 
difficulty  in  water,  being  dissolved  by  2454  parts  water  at  20°  C.  and  154 
parts  boiling  water,  separating,  however,  as  tufts  of  needles  on  cooling. 
It  dissolves  more  easily  in  the  presence  of  alkalies,  ammonia,  or  a  mineral 
acid.  It  is  difficultly  soluble  in  acetic  acid.  Crystals  of  tyrosine  separate 
from  an  ammoniacal  solution  on  the  spontaneous  evaporation  of  the  am- 
monia. One  hundred  parts  glacial  acetic  acid  dissolve  on  boiling  only  0.18 
parts  tyrosine,  and  by  this  means,  especially  on  adding  an  equal  volume  of 
alcohol  before  boiling,  the  leucine  can  be  quantitatively  separated  from  the 
tyrosine  (Habermann  and  Ehrenfeld).  The  /-tyrosine  ethyl  ester  crys- 
tallizes in  colorless  prisms  which  melt  at  108-109°  C.  The  naphthyliso- 
cyanate-Z-tyrosine  melts  at  205-206°.  Tyrosine  can  be  oxidized  with  the 
formation  of  dark-colored  products  by  various  plant  as  well  as  animal 
oxidases,  so-called  tyrosinases  (see  Chapter  I).  By  the  enzyme  occurring 
in  beet-juice  tyrosine  can  be  converted  into  homogentisic  acid  (Gonner- 
MANN  ■*).  Tyrosin  is  identified  by  its  crystalline  form  and  by  the  following 
reactions: 

Piria's  Test.  Tyrosine  is  dissolved  in  concentrated  sulphuric  acid  by 
the  aid  of  heat,  by  which  tyrosine-sulphuric  acid  is  formed;  it  is  allowed  to 
cool,  diluted  with  water,  neutralized  by  BaCOs,  and  filtered.  On  the  addi- 
tion of  a  solution  of  ferric  chloride  the  filtrate  gives  a  beautiful  violet  color; 

'  Erlenmeyer  and  Li])p,  Ber.  d.  d.  chem.  Gesellsch.,  15;  Erlenmeyer  and  Halsey, 
ibid.,  30. 

'  Ibid.,  17. 

^  See  Hoppe-Seyler-Thierfelder,  Handb.  d.  physiol.  u.  pathol.  chem.  Analyse,  7. 
Auflage,  190.3.  Also  E.  Fischer,  Ber.  d.  d.  chem.  Gesellsch.,  32;  Schulze  and  Winter- 
stein,  Zeitschr.  f.  physiol.  Chem.,  45. 

^Pfliiger's  Arch.,  82. 


PHENYLALANINE.  91 

This  reaction  is  disturbed  by  the  presence  of  free  mineral  acids  and  by  the 
addition  of  too  much  ferric  chloride. 

Hofmann's  Test.  If  some  water  is  poured  on  a  small  quantity  of 
tyrosine  in  a  test-tube  and  a  few  drops  of  JMillon's  reagent  added  and  then 
the  mixture  boiled  for  some  time,  the  liquid  becomes  a  beautiful  red  and 
then  yields  a  red  precipitate.  Mercuric  nitrate  may  first  be  added,  then, 
after  this  has  boiled,  nitric  acid  containing  some  nitrous  acid. 

Deniges'  Test,  modified  by  C.  Morner,i  is  performed  as  follows:  To 
a  few  cubic  centimetres  of  a  solution  consisting  of  1  vol.  formaline,  45  vols, 
water,  and  55  vols,  concentrated  sulphuric  acid  add  a  little  tyrosine  in  sub- 
stance or  in  solution  and  heat  to  boiling.  A  beautiful  permanent  green 
coloration  is  obtained. 

CH2.C6H5 

Phenylalanine  (phenyl-a-aminopropionic  acid),  C9HnN02=  CH(NH2), 

COOH 
was  first  found  by  E.  Schulze  and  Barbieri  2  in  etiolated  lupin  sprouts. 
It  is  produced  in  the  acid  cleavage  of  protein  substances.  E.  Fischer 
and  his  collaborators  ^  obtained  3.38  per  cent  phenylalanine  from  haemo- 
globin, 3.0  per  cent  from  horn  substance,  2.5  per  cent  from  ovalbumin  and 
casein,  1.5  per  cent  from  fibroin,  and  0.4  per  cent  from  gelatine.  Abder- 
HALDEN  and  ScHiTTENHELM  obtained  3.89  per  cent  from  elastin. 

The  /-phenylalanine  crystallizes  in  small,  shining  leaves  or  fine  needles 
which  are  rather  difficultly  soluble  in  cold  water  but  readily  soluble  in 
hot  water.  A  5  per  cent  solution  acidified  with  hydrochloric  acid  or  sul- 
phuric acid  is  precipitated  by  phosphotungstic  acid,  while  a  more  dilute 
solution  is  not  precipitated.  On  putrefaction,  phenylalanine  yields  phenyl- 
acetie  acid.  On  heating  with  potassium  dichromate  and  sulphuric  acid 
(25  per  cent)  an  odor  of  phenylacetaldehyde  is  produced  and  benzoic  acid 
is  formed. 

The  separation  and  preparation  of  the  three  amino-acids,  aspartic 
acid,  glutamic  acid,  and  tyrosine,  from  a  mixture  of  hydrolytic  decomposi- 
tion products  of  protein  substances  is  performed  essentially  according 
to  the  method  suggested  by  Hlasiwetz  and  Habermann  with  the  modi- 
fications and  changes  proposed  by  other  investigators.  The  isolation  and 
purification  of  the  amino-acids  can  be  best  accomplished  according  to  E. 
Fischer's  method,  which  consists  essentially  in  esterifying  the  acids  first 
with  hydrochloric  acid  and  alcohol,  separating  the  esters  from  their  hydro- 
chloride by  means  of  alkali,  and  then  fractionally  distilling  the  esters  mider 
very  low  pressure,  and  finally  saponifying  the  different  fractions  by 
boiling  with  water  or  by  heating  with  baryta-water.  It  is  not  ^^•ithin  the 
scope  of  this  book  to  give  a  detailed  description  of  these  methods,  there- 


^  Deniges,  Compt.  rend.,  130;  C.  Morner,  Zeitschr,  f.  physiol.  Chem.,  37. 
^  Ber.  d.  d.  chem.  Gesellsch.,  14,  and  Zeitschr.  f.  physiol.  Chem.,  12. 
^  See  foot-note  1,  p.  84. 


92  THE  PROTEIN  SUBSTANCES. 

fore  we  must  refer  for  further  information  to  Hoppe-Seyler-Thierfel- 
der's  "  Handbuch  der  physiologisch-  unci  pathologisch-chemischen  Analyse,'^ 
7.  Auflage.  and  to  Fischer's  ^  collected  works  on  this  subject. 

We  must  here  add  that  the  preparation  of  the  /?-naphthalenesulpho- 
derivatives  according  to  Fischer  and  Bergell,  of  the  4-nitrotoluenc-2- 
sulpho-compounds  according  to  Siegfried,  and  of  the  a-naphthyliso- 
cyanate  compounds  according  to  Neuberg  and  Manasse^  are  also  of 
importance  in  the  detection  and  isolation  of  many  of  the  amino-acids. 

Cystine,  C6H12N2S2O4  (the  disulphide  of  a'-amino-,5-thiolactic  acid), 
CH2— S— S— CHo 

CH(NH2)  CH(NH2),  was  first  obtained  with  positiveness  as  a  cleavage 
COOH  COOH 

product  of  protein  substances  by  K.  Morxer,  and  then  also  by  Embden. 
KtJLz  3  obtained  it  also  once  as  a  product  of  tryptic  digestion  of  fibrin. 
Morner  obtained  6.8  per  cent  cystine  from  ox-horn,  13.92  per  cent  from 
human  hair,  7.62  per  cent  from  the  membrane  of  the  hen  egg,  2.53  per 
cent  from  seralbumin,  1.51  per  cent  from  serglobulin,  1.17  per  cent  from 
fibrinogen,  and  0.29  per  cent  from  ovalbumin. 

According  to  Neuberg  and  Mayer  ^  two  kinds  of  cystine  occur  in  nature, 
namelv,  stone-cystine  and  protein-cystine.     Stone-cystine  is  the  disulphide  of 

CH2NH2        CH2NH2 
/S-amino-a-thiolactic  acid,  CH — S — S — CH 

COOH  COOH 

It  is  difficult  to  say  which  cystine  we  have  had  in  the  various  cases 
where  it  has  been  found.  The  protein-cystine  has  been  chiefly  obtained 
from  the  protein  substance,  but  also  from  calculi,  while  the  stone-cystine  has 
only  been  obtained  from  urinary  calculi.  Rothera  could  not  find  any 
difference  between  the  stone-cystine  and  the  cystine  prepared  from  hair,  and 
Fischer  and  Suzuki  °  arrived  at  similar  results,  which  seems  to  place  the 
existence  of  stone-cystine  in  doubt.  The  occurrence  of  two  stereoisomeric 
cystines  is  not  improbable,  and  important  observations  of  ^Iorxer  show  that 
the  cystine-yielding  group  of  the  protein  substances  contains  two  cystines. 

Cystine  occurs  in  rare  cases  in  the  urine  or  as  a  calculus,  and  has  also 
been  found  in  ox-kidneys,  in  the  liver  of  the  horse  and  dolphin,  and  in 
traces  in  the  liver  of  a  drunkard.     Abderhaldex^  has  found  cvstine  in 


'  Ber.  d.  d.  chem.  Gesellsch.,  39,  p.  530.  His  collected  Avorks  on  this  subject 
may  be  found  in  Fischer's  "  Untersuchungen  iiber  Amino. Lauren,  PoI\'peptide  und  Pro- 
teine  1899-1900,"  Berlin,  1906. 

^  Fischer  and  Bergell,  Ber.  d.  d.  chem.  Gesellsch.,  35;  Neuberg  and  Manasse,  ibid., 
38;   Siegfried,  Zeitschr.  f.  physiol.  Chem.,  43. 

K.  Morner,  ilrul.,  28,  34,  and  42;  Embden,  ibid.,  32;  Kiilz,  Zeitschr.  f.  Biologic,  27. 

*  Zeitschr.  f.  physiol.  Chem.,  44. 

*  Rothera,  Journ.  of  Physiol.,  32;  Fischer  and  Suzuki,  Zeitschr.  f.  physiol.  Chem.,  45. 

*  Zeitschr.  f.  physiol.  Chem.,  38. 


CYSTINE.  93 

the  urine  and  also  alnindantly  in  the  organs  (spleen)  in  a  case  of  parental 
cystine  diathesis. 

The  constitution  of  cystine  has  Ijeen  explained  by  Friedmaxx.^  and  he 
has  also  established  the  relationship  between  cystine  and  taurine.  Cystine 
is  the  disulphide  of  cysteine,  which  is  a-amino-.9-thiolactic  acid.  From 
cysteine  Friedmaxx  obtained  cysteinic  acid  (aminosulphopropionic  acid), 

CHoSOoOH 
C3H7XS05  =  CH(XH2).  from  which  taurine  is  produced  by  splitting  off  CO2. 
COOH 

Cystine  has  also  been  prepared  s}-nthetically.  Starting  from  ethyl 
formyl  hippurate.  Erlexmeyer.  Jr..  and  Stoop  first  prepared  the  benzoyl- 
serine  ester,  and  then  with  phosphorus  pentasulphide  they  obtained  the 
benzoylcystine  ester.  On  splitting  the  latter  with  HCl  they  obtained 
cysteine,  and  then  inactiye  cy.stine  on  oxidation.  Gabriel  2  has  also  pre- 
pared an  isocysteine  by  the  cleayage  of  rhodandihydrouracil  with  hydro- 
chloric acid,  and  then  inactiye  cystine  by  the  oxidation  of  this  isocysteine. 

Cystine  crystallizes  in  thin,  colorless,  hexagonal  plates.  It  is  not  soluble 
in  water,  alcohol,  ether,  or  acetic  acid,  but  dissolyes  in  mineral  acids  and 
oxalic  acid.  It  is  also  soluble  in  alkalies  and  ammoni^,  but  not  in  ammo- 
nium carbonate.  Cystine  is  optically  actiye.  being  leyorotator}-.  Morxer 
found  it  to  be  (a)^  =  —224.3°.  On  heating  with  hydrochloric  acid  it 
can,  according  to  Morxer.  be  changed  into  a  modification  crj'staUizing  in 
needles  and  with  a  weaker  leyorotator\-  power,  and  indeed  it  can  be 
changed  into  a  dextrorotatory  modification.  On  heating  with  HCl  to 
165°  for  12-15  hours  Xeuberg  and  Mayer  obtained  inactiye  cystine.  It  is 
questionable  whether  this  is  identical  with  the  inactiye  cystine  prepared 
by  Erlexmeyer  s^-nthetically.  By  fungus  fermentation  with  Aspergillus 
niger  they  obtained  dextrorotatory-  cystine.  Cystine  has  no  melting-point 
but  slowly  decomposes  at  258-261°.  On  boiling  cystine  with  caustic  alkali 
it  decomposes  and  yields  alkali  sulphide,  which  can  be  detected  by  lead 
acetate  or  sodium  nitroprusside.  According  to  Morxer  75  per  cent  of 
the  total  sulphur  is  separated.  On  treatment  of  cystine  with  tin  and  hydro- 
chloric acid  it  deyelops  only  a  little  sulphuretted  hydrogen,  and  is  con- 
yerted  into  cysteine.  On  shaking  a  solution  of  cystine  in  an  excess  of  sodmm 
hydrate  with  benzoyl  chloride,  a  yoluminous  precipitate  of  benzoyl  cystine 
is  obtained  (Baumaxx  and  Goldmaxx^).  The  benzoyl  compoimd  melts 
at  182-184°.  The  phenylcyanate  compoimd  melts  at  160°  and  on  boiling 
with  25  per  cent  HCl  is  transformed  into  its  anhydride,  a  hydantoin  melting 
at  119°.  Cystine  forms  crystalline  salts  with  mineral  acids  and  with  bases. 
For  isolating  and  separating  cystine  the  precipitation  with  mercuric  acetate 

'  Hofmeister "s  Beitrage,  3.  p.  1. 

-  Erienmeyer  and  Stoop,  Ber.  d    d.  chem.  Gesellsch.,  36;    Gabriel,  ibiJ.,  38. 

'  Morner,  Zeitschr.  f.  physiol.  Chem.,  34;    Baumann  and  Goldmann,  ibid.,  12. 


94  THE  PROTEIN  SUBSTANCES. 

is  especially  suited.  On  heating  upon  platinum-foil  cystine  does  not  melt, 
but  ignites  and  burns  with  a  bluish-green  flame,  with  the  generation  of  a 
peculiar  sharp  odor.  When  warmed  with  nitric  acid  it  dissolves  with 
decomposition  and  leaves  on  evaporation  a  reddish-brown  residue,  which 
does  not  give  the  murexid  test.  Cystine  is  gradually  precipitated  from  its 
sulphuric  acid  solution  by  phosphotungstic  acid. 

Stone-cystine,  according  to  Neuberg  and  ^Mayer,  differs  in  many  regards 
from  the  ordinary  cystine,  among  which  the  following  will  be  mentioned: 
The  optically  active  stone-cystine  crj^stallizes  in  needles,  the  specific  rota- 
tion is  (q:)d  =  -206°;  it  melts  at  190-192°  with  marked  swelling  up.  The 
benzoyl  compound  melts  at  157-159°;  the  phenylcyanate  compound  melts 
at  170-172°,  and  it  is  not  changed  on  boiling  with  hydrochloric  acid. 

In  the  detection  and  identification  of  cystine  we  make  use  of  the  crystal- 
line form,  the  behavior  on  heating  on  platinum-foil,  and  the  sulphur  reac- 
tion after  boiling  with  alkali.  As  to  its  preparation  from  protein  substances 
see  K.  MoRNER.i  In  regard  to  the  detection  of  cystine  in  the  urine  see 
Chapter  XV. 

CH2.SH 

Cysteine  (a'-amino-,/?-thiolacticacid),C3H7NS02=CH(NH2),  is  formed  from  cys- 

COOH  " 
tine  by  reduction  with  tin  and  hydrochloric  acid.  It  is  also  produced  in  the  cleavage 
of  protein  substances,  but  this  is  considered  by  Morner  and  Patten  ^  as  a  second- 
ary formation,  while  Embden  considers  it  as  primary  from  proteins  poor  in  sul- 
phur. Besides  a-amino-,5-thiocysteine  occurring  in  the  proteins  we  may  prob- 
ably also  have  a  /?-amino-«-thiocysteine.  According  to  Morner  the  thiolactic  acid 
which  he  obtained  on  the  decomposition  of  cystine  probably  originates  from  the 
latter,  while  the  a-amino-;?-thio cysteine  is  probably  the  mother-substance  of  the 
alanine  obtained  at  the  same  time.     Cysteine  can  be  readily  converted  into  cj'stine. 

Towards  alkalies  and  lead  acetate  it  acts  like  cystine.  With  sodium  nitro- 
prusside  and  alkali  it  gives  a  deep  purple-red  coloration ;  with  ferric  chloride  the 
solution  gives  an  indigo-blue  coloration  which  quickly  disappears. 

CH3 

Thiolactic  acid  (a-thiolactic  acid),  C3HtiS02=  CH.SH,  has  been  found  once  as  a 

COOH 
cleavage  product  of  ox-horn  by  Baumann  and  Suter.  It  has  been  shown  by 
Friedmann  that  this  acid  is  a  regular  cleavage  product  of  keratin  substances,  and 
that  it  can  also  be  obtained  from  the  proteins.  Frankel  ^  obtained  the  acid 
from  haemoglobin.  The  pyroracemic  acid  obtained  by  Morner  as  a  decomposi- 
tion product  from  several  protein  substances  originates,  according  to  Morner, 
only  in  part  from  the  cystine. 

CTT    NTT-T 

Taurine*  (aminoethylsulphonic  acid),  C2H7NS03  =  ,v„^' ,^  ^r^xj-  ^^^  ^lot 

Crl2.k)U2.Uxl 

boen  obtained  as  a  cleavage  product  of  protein  substances;    still  its  origin 


'  Zeitschr.  f.  physiol.  Chem.,  34. 

2  See  foot-note  2,  p.  28. 

'Suter,  Zeitschr.  f.  physiol.  Chem.,  20;  Friedmann,  Hofmeister's  Beitrage,  3; 
Frankel,  Sitzungsber.  d.  Wicn.  Akad.,  112,  II,  b,  1903. 

*  Taurine  does  not  belong  to  the  cleavage  products  of  the  proteins,  but  for  practical 
reasons  it  will  be  described   in  connection  with  cvstine. 


TAURINE.  95 

from  proteins  has  been  shown  by  Friedmann  by  the  close  relationship 
that  taurine  bears  to  cysteine.  Taurine  is  especially  known  as  a  cleavage 
product  of  taurocholic  acid  and  may  occur  to  a  slight  extent  in  the  intestinal 
contents.  Taurine  has  also  been  found  in  the  lungs  and  kidneys  of  oxen 
and  in  the  blood  and  muscles  of  cold-blooded  animals. 

Taurine  crystallizes  in  colorless,  often  in  large,  shining,  4-  or  6-sided  prisms. 
It  dissolves  in  15-16  parts  of  water  at  ordinary  temperatures,  but  rather 
more  easily  in  warm  water.  It  is  insoluble  in  absolute  alcohol  and  ether; 
in  cold  spirits  of  wine  it  dissolves  slightly,  but  better  when  warm.  Taurine 
yields  acetic  and  sulphurous  acids,  but  no  alkali  sulphides,  on  boiling  with 
strong  caustic  alkali.  The  content  of  sulphur  can  be  determined  as  sul- 
phuric acid  after  fusing  with  saltpetre  and  soda.  Taurine  combines  with 
metallic  oxides.  The  combination  with  mercuric  oxide  is  white,  insoluble, 
and  is  formed  when  a  solution  of  taurine  is  boiled  with  freshly  precipitated 
mercuric  oxide  (J.  Lang  ^).  This  compound  may  be  used  in  detecting  the 
presence  of  taurine.     Taurine  is  not  precipitated  by  metallic  salts. 

The  preparation  of  taurine  from  ox-bile  is  very  simple.  The  bile  is  boiled 
a  few  hours  with  hydrochloric  acid.  The  filtrate  from  the  dyslysin  and 
choloidic  acid  is  concentrated  well  on  the  water-bath,  and  filtered  hot  so 
as  to  remove  the  common  salt  and  other  substances  which  have  separated. 
The  solution  is  evaporated  to  dryness  and  the  residue  dissolved  in  5  per 
cent  hydrochloric  acid,  and  precipitated  with  10  vols.  95  per  cent  alcohol. 
The  crystals  are  readily  purified  by  recrystallization  from  water.  The 
alcoholic  solution  can  be  used  for  the  preparation  of  glycocolL  After  the 
evaporation  of  the  alcohol,  the  residue  is  dissolved  in  water,  treated  with  a 
solution  of  lead  hydroxide,  filtered,  the  lead  removed  by  H2S,  and  the  filtrate 
strongly  concentrated.  The  crystals  which  separate  are  dissolved  and  de- 
colorized by  animal  charcoal  and  the  solution  then  evaporated  to  crystalli- 
zation. 

Though  taurine  shows  no  positive  reactions,  it  is  chiefly  identified  by 
its  crystalline  form,  by  its  solubility  in  water  and  insolubility  in  alcohol,  by 
its  combination  with  mercuric  oxide,  by  its  non-precipitability  by  metallic 
salts,  and  above  all  by  its  sulphur  content. 

Oxymonamino-acids. 

CH2(0H) 
Serine  (a-amino-^^-oxypropionic  acid),C3H7N03  =  CH(NH2),  was  obtained 

COOH 
by  E.  Fischer  and  his  collaborators  2  as  a  cleavage  product  from  fibroin  (1.6 
per  cent),  horn  substance  (0.68  per  cent),  sericine,  gelatine  (0.4  percent), 
and  casein.     Kossel  and  Dakin  ^  obtained  7.8  per  cent  from  salmine. 
Synthetically  it  was  prepared  by  E.  Fischer  and  Leuchs  *  from  ammonia. 


'  See  Maly's  Jahresber.,  6. 

^  See  foot-note  1,  p.  84. 

'  Zeitsehr.  f.  physiol.  Chem.,  41. 

''  Ber.  d.  d.  chem.  Gesellsch.,  35,  and  Sitzungsber.  d.  Akad.  d.  Wiss.,  Berlin,  1902- 


96  THE  PROTEIN  SUBSTANCES. 

hydrocyanic  acid,  and  glycolyl  aldehyde.  Serine  has  also  been  prepared  by 
Erlenmeyer,  Jr.,  and  Stoop  ^  by  starting  with  ethyl  formyl  hippurate 
and  converting  it  by  reduction  into  benzoylserine  ester,  from  which 
benzoylserine  was  obtained  by  saponification  with  alcoholic  potash,  and 
then  from  this,  serine  was  obtained  by  boiling  with  sulphuric  acid. 

Isoserine  (/9-amino-a-oxypropionic  acid)  has  been  prepared  by  Ellinger  from 
<liaminopropionic  hydrobromide  and  silver  nitrite,  and  by  Neuberg  and  Silber- 
MANN  ^  from  diaminopropionic  hydrochloride. 

Serine  does  not  dissolve  readih'  in  cold  water  (23  parts  water  at  20°  C), 
but  more  easily  in  hot  water.  The  solution  is  inactive  and  has  a  sweet 
taste.  Serine  crystallizes  from  water  in  thin  plates,  which  melt  at  240° 
with  the  generation  of  a  gas. 

According  to  Skraup,  oxyaminosuccinic  acid,  CnHyNOs,  is  very  jorobably  a 
hydrolytic  cleavage  product  of  the  proteins.  This  acid  has  been  prepared  syn- 
thetically by  Neuberg  and  Silbermann  from  diaminosuccinic  acid  and  barium 
nitrite  in  sulphuric-acid  solution.  Oxyaminosuberic  acid,  CgHisNOj,  has  been 
found  by  Wohlgemuth  as  a  cleavage  product  of  a  liver  nucleoproteid,  and  the 
acid  CeHisNOe,  isolated  by  Orgler  and  Neuberg  ^  from  chondroitin-sulphuric 
acid,  but  not  from  protein,  and  considered  by  them  as  tetraoxyaminocaiaroic  acid, 
seems  also  to  belong  to  the  oxyamino-acid  group. 

2.    Diamino-acids  (Hexone  Bases). 
Arginine  (guanidine-a-amino valerianic  acid), 

C6Hi4N402=  (CH2)2  , 

CH(NH2) 
COOH 

first  discovered  by  Schulze  and  Steiger  in  etiolated  lu])in-  and  pumpkin- 
sprouts,  has  later  been  found  in  other  germinating  plants,  in  tubers  and 
roots.  GuLEWiTSCH  has  found  arginine  in  the  ox-spleen.  It  was  first 
found  by  Hedin  as  a  cleavage  product  of  horn  substance,  gelatine,  and 
several  proteins,  and  then  by  Kossel  and  his  pupils  as  a  general  cleav- 
age product  of  protein  substances  as  a  class.  The  greatest  quantity  was 
obtained  from  the  protamines;  but  the  histones  and  certain  plant  proteins 
(edestin  and  the  protein  from  pine  seeds)  also  yield  abundant  arginine. 
Arginine  also  occurs  among  the  products  of  tryptic  digestion  (Kossel  and 
Kutscher). 

On  boiling  with  baryta-water  as  well  as  by  the  action  of  an  enzyme, 

'  Ber.  d.  d.  chem.  Gesellsch.,  35. 

^Ellinger,  ibid.,  87;    Neuberg  and  Silbermann,  ibid.,  37. 

'Skraup,  Zeitschr.  f.  physiol.  Chem.,  42;  Neuberg  and  Silbermann,  ibid.,  44; 
"Wohlgemuth,  ibid.,  44;    Orgler  and  Neuberg,  ibid.,  37. 


ARGIXIXE.  97 

arginase,  discovered  by  Kossel  and  Dakix/  arginine  yields  urea  and 
ornithine.  Arginine  has  been  prepared  s}Tithetically  from  ornithine  (a-o-di- 
aminovalerianic  acid)  and  cyanamide  by  Schulze  and  Wintersteix.^ 

Arginine  cr}'stallizes  in  rosette-like  tufts,  plates,  or  thin  prisms,  is  readily 
soluble  in  water  and  nearly  insoluble  in  alcohol.  With  several  acids  and 
metallic  salts  it  forms  cr\-stalline  salts  and  double  salts  respectively.  Its 
acidified  waters'  solution  is  precipitated  by  phosphotungstic  acid.  The  most 
important  salts  are  the  copper-nitrate  (C6Hi4X402)2.Cu(X03)2+3H20  and 
the  silver  salts  C6H14X4O2.HXO3  +  AgXOs  (the  more  readily  soluble)  and 
C6Hi4X402.AgX03  +  ^H20  (the  more  difficultly  soluble)  and  its  compound 
with  picrolonic  acid  (Steudel^). 

Arginine  is  dextrorotatory-,. but  the  arginine  obtained  Ijy  Kutscher  in 
the  tr}'ptic  digestion  of  fibrin  was  inactive.  On  oxidation  with  perman- 
ganate it  splits  off  guanidine,  which  can  be  precipitated  with  sodium  picrate. 
Orglmeister  ^  bases  his  method  for  the  quantitative  estimation  of  arginine 
in  mixtures  obtained  by  hydrolysis  upon  this  fact. 

(:h2.(xh,) 

Ornithine  (a-5-diaminovalerianic  acid),C5Hi,X202=  (^jj('>^-jj  \,  is  not  a  primary 

COOH  " 
cleavage  product  of  proteins,  but  is  formed  from  arginine  on  boiling  with  barvta- 
water.  Jaffe,^  who  first  discovered  this  body,  obtained  it  as  a  cleavage  product 
from  ornithuric  acid,  which  is  found  in  the  urine  of  hens  fed  with  benzoic  acid. 
The  ornithine  which  E.  Fischer  and  recently  Sorexsex  ^  have  prepared  s}"!!- 
thetically  yields,  as  shown  by  Ellixger,  putrescine  (tetramethylenediamine), 
C4Hg(XH2)2,  on  putrefaction.  A.  Loewy  and  X'euberg  '  have  shown  that  orni- 
thine is  split  into  putrescine  and  CO,  in  the  organism  of  cystinuria  patients. 

OrnitMne  is  a  non-crystalline  substance  which  dissolves  in  water,  giving  an 
alkaline  reaction,  and  yields  several  crystalline  salts.  It  is  precipitated  by 
phosphotungstic  acid  and  several  metallic  salts,  but  not  by  silver  nitrate  and 
baryta-water  (differing  from  arginine).  Ornithine  hydrochloride  is  dextrorotatory ; 
the  synthetically  prepared  is  inactive.  On  shaking  ornithine  with  benzoyl  chloride 
and  caustic  soda  it  is  converted  into  dibenzoylornithine  (ornithuric  acid).  On 
splitting  artificially  prepared  racemic  ornithuric  acid  Sorexsex  has  .sho\\Ti  that 
the  naturally  occurring  ornithuric  acid  is  identical  with  the  dextrorotatory  a-3- 
dibenzoyldiamino valerianic  acid. 

Diaminoacetic  acid,  CoHeX'oO,  =CH(NH2)2COOH,  was  obtained  by  Drechsel* 
as  a  cleavage  product  of  casein  by  boiling  with  tin  and  hydrochloric  acid.     It 

^  Schulze  and  Steiger,  Zeitschr.  f.  physiol.  Chem.,  11;  Schulze  and  Castoro,  ibid.,  41; 
Gulewitsch,  ibid.,  30;  Hedin,  ibid.,  20  and  21;  Kossel  and  Kutscher,  ibid..  22,  25,  26; 
Kossel  and  Dakin,  ibid.,  11. 

2  Ber.  d.  d.  chem.  Gesellsch.,  32,  and  Zeitschr.  f.  physiol.  Chem.,  34. 

'Zeitschr.  f.  physiol.  Chem.,  3"  and  44. 

*  Hofmeister's  Beitrage,  7. 

^  Ber.  d.  d.  chem.  Gesell.sch.,  10  and  11. 

'Fischer,  ibid.,  34;    Sorensen,  Zeitschr.  f-.  physiol.  Chem.,  44. 

'  EUinger,  Zeitschr.  f.  physiol.  Chem.,  29;    Loewy  and  Xeuberg,  ibid.,  43. 

*Ber.  d,  sachs.  Ges.  d.  Wissensch.,  44. 


98  THE  PROTEIN  SUBSTANCES. 

crystallizes  in  prisms  and  gives  a  monobenzoyl  compound  which  is  not  very  soluble 
in  cold  water  and  is  nearly  insoluble  in  alcohol,  and  can  be  used  in  the  isolation 
of  the  acid. 

CH2(NH2) 

Lysine   (a-£-diaminooaproic    acid),  C6Hi4N202=  r;TJVTy-^TT  x-    was    first 

Lrl(jNrl2) 

COOH 
obtained  by  Drechsel  as  a  cleavage  product  of  casein.  Later  he  and  his 
pupils,  as  well  as  Kossel  and  others,  found  it  among  the  cleavage  products 
r)f  various  proteins.  It  has  not  been  detected  in  some  vegetable  pro- 
teins such  as  zein  and  gluten-protein.  E.  Schulze  found  lysine  in  ger- 
minating plants  of  the  Lupinus  luteus,  and  Winterstein  found  it  in  ripe 
cheese.  It  has  been  obtained  in  largest  amounts  (28.8  per  cent)  by  Kossel 
and  Dakin  ^  from  the  protamine  a-cyprinine. 

Lysine  has  been  synthetically  prepared  by  E.  Fischer  and  Weigert.^ 
This  lysine  was  inactive,  w^hile  that  prepared  from  protein  is  always  optic- 
ally active  and  dextrorotatory.  On  heating  with  barium  hydrate  it  is 
converted  into  the  inactive  modification.  According  to  Ellinger^  lysine 
yields  cadaverine  (pentamethylenediamine),  C5HiofNH2)2.  f^n  putrefaction, 
and  this  base  is  formed  from  the  lysine  in  the  organism  of  those  with  cysti- 
nuria  and  at  the  same  time  CO2  is  split  off  (A.  Loewy  and  Neuberg). 

Lysine  is  readily  soluble  in  water  but  is  not  crystalline.  The  aqueous 
solution  is  precipitated  by  phosphotungstic  acid  Init  not  by  silver  nitrate 
and  baryta-water  (differing  from  arginine  and  histidine).  It  gives  two 
hydrochlorides  \vith  hydrochloric  acid,  and  with  platinum  chloride  a 
chloroplatinate  w^hich  is  precipitable  by  alcohol  and  has  the  compo- 
sition C6Hi4N202.H2PtCl6 +C2H5OH.  It  gives  two  silver  salts  with 
AgNOs;  one  has  the  formula  AgN03+C6Hi4N202  and  the  other  AgN03  + 
CeHi4N202.HN03.  With  benzoyl  chloride  and  alkali,  lysine  forms  an  acid, 
lysuric  acid,  C6Hi2(C7H50)2N202  (Drechsel),  which  is  homologous  with 
ornithuric  acid  and  whose  difficultly  soluble  acid  l)arium  salt  may  be  used 
in  the  sejjaration  of  lysine.^  The  rather  insoluble  picrate,  which  is  pre- 
cipitated from  a  not  too  dilute  solution  of  the  hydrochloride  by  sodium 
picrate,  may  also  be  used  in  the  detection  of  lysine. 

'  Drechsel,  Arch.  f.  (Anat.  u.)  Physiol.,  1891,  and  Ber.  d.  d.  chem.  Gesellsch.,  25; 
Siegfried,  Arch.  f.  (Anat.  u.)  Physiol.,  1891,  and  Bor.  d.  d.  chem.  Gesellsch.,  24;  Hedin, 
Zeitschr.  f.  physiol.  Chem.,  21;  Kossel,  ibid.,  25;  Kossel  and  Mathews,  ibid.,  25; 
Kossel  and  Kutscher,  ibid.,  31;  Kutscher,  ibid.,  29;  Schulze,  ibid.,  28;  Winterstein, 
cited  in  Schulze  and  Winterstein,  Ergebnisse  der  Physiologie,  I,  Abt.  1,  1902;  Kossel 
and  Dakin,  Zeitschr.   f.  physiol.  Chem.,  40. 

^  Ber.  d.  d.  chem.  Gesellsch.,  35. 

'  Zeitschr.  f.  physiol.  Chem.,  29. 

•*  Drechsel,  Ber.  d.  d.  chem.  Gesellsch.,  2-S;  .see  also  C.  Willdenow,  Zeitschr.  f. 
physiol.  Chem.,  25. 


« 

HISTIDINE.  99 

KuTSCHER  and  Loiimann  '  have  found  a  lysine  having  somewhat  different 
properties  in  the  final  products  of  pancreas  autolysis. 

Lysatine  or  Lysatinine.  The  formula  of  this  substance  is  either  CeH  gNjO^  or 
CgHiiNsO +Hv;0.  In  the  first  case  this  base  would  appear  to  be  a  homologue  of 
creatine,  C4H6N3O2,  and  in  the  other  case  a  homologue  of  creatinine,  C4H7N3O,  and 
this  is  the  reason  why  this  body  is  called  lysatine  as  well  as  lysatinine.  It  is  still 
a  question  whether  lysatine  is  a  chemical  individual  or,  as  Hedin  suggests,  only 
a  mixture  of  lysine  and  arginine.^ 

Histidine,  C6H9N3O2,  is  perhaps  not  a  diamino-acid,  as  Fraxkel^ 
first  showed,  but,  according  to  the  investigations  of  H.  Pauly,   Kxoop 

CH— NH\ 

C N/^^ 

and  Windaus,^  is  an  a(?)-amino-/?-imidazolpropionic  acid,  CH2 

CHNH2 
COOH 

Fraxkel  ^  has  made  several  objections  to  Pauly,  Knoop  and  Windaus's 
view  that  histidine  is  an  imidazol  derivate,  which  seem  to  be  well  founded, 
therefore  the  question  as  to  the  constitution  of  histidine  remains  still  an 
open  one. 

Histidine  ^  was  first  discovered  by  Kossel  in  the  cleavage  products  of 
sturine.  It  was  then  found  by  Hedin  in  the  cleavage  products  of  pro- 
teins by  acid  hydrolysis,  and  by  Kutscher  among  the  products  of  tryptic 
digestion,  and  finally  also  as  a  cleavage  product  of  diiTerent  protein  sub- 
stances. It  does  not  occur  in  the  protamines,  with  the  exception  of  sturine. 
Of  the  protein  bodies  globin  (from  horse-haemoglobin)  seems  to  be  richest  in 
histidine,  as  Abderhalden  found  10.96  per  cent.  It  also  occurs  in  germi- 
nating plants  (E.  ScHULZE^). 

Histidine  crystallizes  in  colorless  needles  and  plates  and  is  readily  soluble 
in  water,  but  less  soluble  in  alcohol,  and  has  an  alkaline  reaction.  It 
is  precipitated  by  phosphotungstic  acid,  but  this  precipitate  is  soluble 
in  an  excess  of  the  precipitant  (Frankel).  With  silver  nitrate  alone  the 
aqueous  solution  is  not  precipitated;  on  the  careful  addition  of  ammonia 
or  baryta-water  an  amorphous  precipitate,  which  is  readily  soluble  in 
an  excess  of  ammonia,  is  obtained.     Histidine  can  be  precipitated  ])v  mr;- 


'  Zeitschr.  f.  physiol.  Chem.,  41. 

^  Hedin,  ihiil.,  21;  Siegfried,  ibid.,  35. 

3  Sitzunosber.  d.  Wien.  Akad.,  112,  II,  b,  190.3. 

''  Pauly,  Zeitschr.  f.  physiol.  Chem.,  42;  Knoop  and  Windaus,  Hofmeister's  Bci- 
trilge,  7. 

^  Hofmeister's  Beitrage,  8. 

'  As  histidine  is  always  ol)tained  with  the  diamino-acids  it  is  called  a  hexone  base, 
hence  it  will  be  treated  here  with  the  diamino-acids. 

'  Kossel,  Zeitschr.  f.  physiol.  Chem.,  22;  Hedin,  ibid.,  Kutscher,  ibid.,  25;  Wetzel, 
ibid.,  20;  Lawrow,  ibid.,  28,  and  Ber.  d.  d.  chem.  Gesellsch.,  34;  Kossel  and  Kutscher, 
Zeitschr.  f.  physiol.  Chem.,  31;  Hart,  ibid.,  33;  Abderhalden,  ibid.,  37;  Schulze,  ibid., 
24  and  28. 


lOO  TEIE  PRCVTEIS:  SITR^TAATES. 

cumic  fWksmfe^  or^  sItiM  belrter,  by  tlte  swIfp'&Latie  andtfi!e<i  witii  sulphuric  acid, 
amd  eaaa  ia  tlafe  tray  be  sep^iateti  frojaat  th©  tltammo^cids  as  well  as  from 
Ijne  inoiiiaiimiDi«>-a(ekfe  (Kosjel  and  Pattex).  The  Ii>-drocMoride  cn-stal- 
Ifiaes;  III  Ibeawritiffiaill  plate?  (,R\rEE).  dissolves  ratker  readily  in  water,  but  is 
iBBseJhfflfete  BBi  afedlMji  aini«J  et!i:er>  "Wifelt  !mii:oeMoirt<j  ajftd.  aad  metliyl  akohol 
•  :.  -  :1ft  «MiiyT!fcro«W®rtde  of  kistldinje  metbLyl  ester.  wMch.  melts  at  1%"^. 
v.  -  -i'  is  tevenrodatiiMnr,,  while  its  solutitm  in  hydrocMortc  acid  is  dextro- 
KjiftaltocT.    Qtt  ■wramniMinig  it  ^ve?  the  bitiret  test  (HEsaoG  ^).  and  it  also  gi\'es 

"'  '^  ••■■  "■        " ed  as  su^:^:ested  by  Feschee  (,st^e  Xanthine. 

i  giv\es  a  "very  beautiful  diazonreaction  with 

;<ailpjho£twe  a«fM  m  soiutkuois  made  atkaEne  with  sodium  carbon- 
vjig  t©  Paitly  is  deep  chern.'-red  in  dilutions  of  1:20  000 
;.       .  V  red  im  1:100  000'  (tyrosine  gtv-es  a  similar  reaction). 

■••.^tt«i-«L  of  the  abo've  basses  -we  can  first  precipitate  all  the 
Ijsjus^tj,  t  -  ".i^tic  acid,  w-hen  the  monamino-acids  remain  in  solu- 

tk«iu    T  e  is^  dewimpose^-i  in  boiling  water  by  barium  hyi.irate 

amd  tht"       -  -       :amed  as  sih"er  compounds  from  this  tiltrate.     In  regard 
tO'  furth^;  ......~^  we  must  refer  to  the  woifcj  of  D-rechsejl  and  Hkdlx 

aUre^ady  cited.    KiotssasiL  and  KrcscHBua  and  recently  Wixteksthiix  -  have 
<•-.;-•  •^,,-,1  a  method  of  sepaorating  histidiiae  and  arginine  sus  sil\-er  compoimct? 
-:-ae'.  and KosS'EL. and  Pattbix  have  proposed  a  method  of  separating 
hijCiUiae  fftofln  argintne  by  means  of  mercuric  sulphate. 

We  give  betow  a  tabulation  of  the  antjounts  of  the  three  hexone  basses 
iodaiBttiii  in  cectain  piiro>tein  substamj^es  (in  weight  per  cent) : 

Arpxiiae  Lysine  Histidine 

SfttEmH?*      5!v.:i  12.0  12. d 

CNcpctniae^  ^-JB'^ 4.^  3J<.{^  0.0' 

4L^b«- p.w«3imin«es  * 62-5 — ST.  4  0'.  ©  00 

Hkfiyasfs*. 14.315— 15.K  1.7— S. 3  1.21—2.34 

C^»titt*. 4.Il)--4.S4  l.ftJ— 5.JJ0  2.53-^2.59 

SSfTBtewiiEa!  ('ftens!  BBoeailt)!*.  ....... . 5. 0<§  52!$  2.66 

Hirt.«etgni<K.Okii«  *. . S.5S  3.06— T.0S  0.3^-1.12 

P^wftuxs^^niitHijiBWtie' *..,............ .          455  3  OS  3. 35 

E&sftBBi *.  ...-.■. 11.0^—14.07  1.3  1.17 

FwteM  ftwm  WNSsSfesat  seed* '. ms-— 11.3;  0.25— 0.7^  0.(52— 0.7S 

CStelwa.  «taB«£ii ' 44  2.15  1.16 

IG&alWffi  Mw*««ns  *. 2-75 — 3;..13:  00  0.43 — 1.5S 

Grih*£Bif»aaiii*.... T.«Ki— 9i.S  2.4!J— «.0  0  40 

Ofcstecnt*.. 0  3  -t-  0  027 

"^  Ktfcsswii  aoui  Fitfititicu  Ziiit^br..  L  p&jjnsajL  ChKCU..,  3J>;    Bauer.  iJbid..  22;    Hensug, 

-  KotssfJ  ami  KoittsvJiBir,  iAa£..  Si.;.  Wtatersfeeoi.  iM£^^  -IS,";  Koisel  and  Pafetea,  L  G-. 

*  Koisail:  ami  Ktrtscfter.  Z&ifiistf&ir.  ff.  p&ijrsajL  C&mul..  SL 
*H!art.  ii6«i£..  SL 

*  5s«f&db8  ami  Wtoiwcstieai,  e6»i.^  3S;    see  aLsci  Kocsssel,  Ber.  d.  d.  chem.  CteseBiseh., 

ai,?2a5^ 

*  Bv^-iiisell  ami  Ktxb^&er.  2^s«i&r.  fi.  p&jjrsbL  Chem..  2S»,  ami  Richards  ami  Gies, 
AnjBtr.  Jbairtii.  <<?£  PfecyswL..  T. 

^  Koesfll  ami  PtiMa.  2ufi.t:<«t&ir.  f.  phystpL  Cbem..  -Ml 


« -PROLINE.  101 


Oxydiamino-acids. 


Oxydiaminosebacic  acid,  C,„H.,,N.,05,  has  been  isolated  as  a  copper  salt  by 
WoHi-ciEMi'TH  '  from  a  iiueleoproteid  oi  the  liver.  The  free  acid  was  obtained 
as  small  white  plates.  It  is  soluble  with  diftieulty  in  hot  water,  insoluble  in  cold 
water  and  in  aleohul.  It  was  optically  inactive  in  hydrochloric  acid.  The 
beautifully  crystalline  phenylcyanate  compound  had  a  meltitifj-puint  of  200'"\ 

lyiox  yd  Laminate  id  ni-ir  acid,  C\Hi,,N,;0,„  has  been  obtained  by  Skkaup  -  on  the 
hydrolysis  of  casein  with  hydrt)chloric  acid.  The  copper  salt  crystallizes  in  beauti- 
ful deep  bluish-violet  rosettes  which  are  composed  of  long,  irregular,  right- 
angled  plates.  It  is  cjuite  soluble  in  cold  water.  The  free  acid  crystallizes  iu 
fern-like  formations.  Besides  this  acid  Skkaip  obtained  two  other  acids  which 
he  calls  cascanic  acid,  C„H,uN^,C)7,  and  caseirdc  acid,  Civll.'-tN-Os.  The  caseanic  acid 
crystallizes,  melts  at  190-191",  is  tribasic,  and  is  probably  an  oxydiamino-acid. 
The  caseinic  acid  is  tlibasic  and  occurs  in  two  modifications.  The  one  which 
melts  at  228''  is  faintly  dextrorotatory;  the  other  modification  melts  at  245" 
and  is  optically  inactive.  Both  crystallize,  but  the  inactive  form  does  not  yield 
well-ileflned  crystals.     Caseinic  acid  seems  also  to  be  an  oxydiamino-acid. 

Diaminotrioxydudccarioic  acid,  Vy.li._^^.Oi,  is  an  acid  obtained  by  Fischkk  and 
.\BnKKnALOEN  ^  Oil  the  hydrolysis  of  cast>iu  and  seems  to  stand  close  to  Skr^vup's 
caseinic  acid,  but  ditTers  from  it  in  its  optical  properties.  This  acid  is  fauitly 
levorotatory :  (ci:)p  =-  about  —9°.  It  crystallizes  in  plates,  which  grow  into  rosettes 
or  spherical  aggregations.  It  has  a  faint  bitter  taste,  gives  a  crystalline  hydro- 
chloride which  is  slightly  soluble  in  strong  hydrochloric  acid,  and  gives  a 
crystalline  copper  salt. 

3.    Pyrrol  and  Indol  Derivatives. 
«-Pyrrolidine-carboxylic  acid,  abbreviated  to  tt-ProUne,  C5II9NO2, 

I  I 

CH3   CH.COOH, 


Ml 

was  prepared  by  E.  Fischkk  as  a  cleavage  product  from  casein  (3.2  per 
cent)  and  ovulbutnin  (1.55  per  cent),  and  by  him  and  his  collaborators  in 
the  tryptic  digestion  of  casein,  and  as  a  cleavage  product  of  haemoglobin 
(1.46  per  cent),  gelatine  (5.2  per  cent),  horn  substance  (3.60  per  cent),  and 
from  silk  fibroin.^  The  acid  thus  obtained  was  generally  the  levorotatory 
moditicatioii.  Kosskl  and  Dakin  ^  obtained  11  i)er  cent  a-proline  from 
salmine,  while  Abdkrhalden  and  his  co-workers*^  obtained  2.25  per  cent 
from  ovalbumin,  1.46  from  thymus  hist  one,  1.74  from  elasthi,  and  3.4-3.5 
per  cent  from  keratin  substances.     a-ProUne    also  occurs    in    scombrine 


'  Ber.  d.  d.  chem.  Cesellsch.,  37,  and  Zeitschr.  f.  physiol.  Chem.,  44. 

•  Zeitschr.  f.  physiol.  Choni.,  42. 
'  Ibid. 

*  Fischer,  ibv.1.,  33  and  3o.     >See  also  foot-note   I,  p.  84. 
'  Zeitschr.  f.  physiol.  Chem.,  41. 

'  Abderhalden  and  I'regl,  ibid.,  46;  with  Rona,  ibid.,  41;  with  Schittenhelni,  ibid., 
41;  with  Wells  and  Le  Count,  ibid.,  40. 


102  THE  PROTEIN  SUBSTANCES. 

and  clupeine,  but  not  in  sturine,  which  according  to  Kossel  seems  to 
contradict  the  view  as  to  the  common  origin  of  ornithine  and  a-proline. 

SORENSEN  1  by  means  of  a  general  method  of  preparing  a-amino-acids 
synthetically  has  prepared  a-amino-o-oxyvalerianic  acid  from  phthalimide- 
malonic  ester  and  has  obtained  a-proline  from  this  by  evaporating  with 
hydrochloric  acid,  at  the  same  time  splitting  off  water. 

This  acid  is  readily  soluble  in  water  and  alcohol  and  crystallizes  in  flat 
needles  which  melt  at  203-206°  C.  with  an  odor  of  pyrrolidine.  The  solu- 
tion acidified  with  sulphuric  acid  is  precipitated  by  phosphotungstic  acid. 
In  the  detection  of  this  acid  we  make  use  of  the  copper  salt,  the  anhy- 
dride of  the  phenylisocyanate  compound  (melting-point  144°),  and  the 
picrate  (Alexandroff^).  The  inactive  acid  and  its  compounds  show  some- 
what varying  properties.  In  regard  to  the  detection  of  this  acid  we  refer 
to  p.  91. 

In  the  hydrolysis  of  gelatine  and  casein  E.  Fischer  '  obtained  an  amino- 
acid  having  the  formula  CjHgNOg,  which  on  reduction  yielded  a-pyrrolidine-carbo- 
xylic  acid,  and  which  according  to  Fischer  is  an  oxypyrrolidine-a-carboxylic 
acid.     Leuchs  ^  has  synthetically  prepared  two  similar,  inactive  acids. 

Indolaminopropionic  acid  (tryptophane,  proteinochromogen),CiiHi2N202, 
C.CH2.CH(NH2)COOH  C.CH(NH2).CH2.Co6h 

C6H4/VH  or    C6H4<^CH 

NH  NH 

is  one  of  the  cleavage  products  of  the  proteins  formed  in  tryptic  digestion 
and  other  deep  decompositions  of  the  proteins,  such  as  putrefaction,  cleavage 
with  baryta-water  or  sulphuric  acid.  It  gives  a  reddish -violet  product  with 
chlorine  or  bromine  which  is  called  proteinochrome.  Nencki  ^  considered 
tryptophane,  which  name  is  generally  given  to  this  acid,  as  the  mother- 
substance  of  various  animal  pigments. 

Trvptophane  was  first  prepared  in  a  pure  form  by  Hopkins  and  Cole,® 
and  they  considered  it  as  skatolaminoacetic  acid.  After  Ellinger^  showed 
that  skatolcarbonic  acid  (Salkowski)  and  skatolacetic  acid  (Nencki) 
were  indolacetic  acid  and  indolpropionic  acid  respectively,  we  have  con- 
sidered tryptophane  as  indolaminopropionic  acid. 

■  Zeitschr.  f.  physiol.  Chern.,  44. 

^  In  regard  to  the  preparation  of  the  phenylisocyanate  compounds  of  the  amino- 
acids,  see  Paal,  Ber.  d.  d.  chem.  Gesellsch.,  27;  Mouneyrat,  ibid.,  33,  and  Hoppe- 
Seyler-Thierfelder's  Handbuch,  7.  Aufl.;  Alexandroff,  Zeitschr.  f.  physiol.  Chem.,  46. 

'  Ber.  d.  d.  chem.  Gesellsch.,  35  and  86. 

5  In  regard  to  trytophano,  sec  Stadelmann,  Zeitschr,  f.  Biologie,  26;  Neumeister, 
ibid.,  26;  Nencki,  Ber.  d.  d.  chem.  Gesellsch.,  2S;  Beitler,  ibid.,  SI;  Kurajeff,  Zeitschr. 
f.  physiol.  Chem.,  26;    Klug,  Pfliiger's  Arch.,  86. 

'Journ.  of  Physiol.,  27. 

'Ber.  d.  d.  chem.  Gesellsch.,  37  and  38. 


INDOLAMINOPROPIONIC  ACID.  103 

Tryptophane  crystallizes  in  shining  plates  which  are  readily  soluble  in  hot 
Avater,  less  soluble  in  cold  water  and  in  alcohol.  On  heating  sufficiently, 
it  yields  indol  and  skatol.  It  gives  the  Adamkiewicz-Hopkins  reaction 
and  a  rose-red  coloration  on  the  addition  of  bromine-water  (tryptophane 
reaction).  If  a  pine  stick  moistened  with  hydrochloric  acid  and  then  washed 
off  be  introduced  into  a  concentrated  tryptophane  solution,  it  becomes 
purple-colored  on  drying  (pyrrol  reaction).  Tryptophane,  as  Hopkins  and 
Cole  ^  shoAved  later,  yields  skatolacetic  acid  (indolpropionic  acid)  on  anae- 
robic putrefaction,  and  skatolcarbonic  acid  (indolacetic  acid),  skatol,  and 
indol  on  aerobic  putrefaction. 

In  regard  to  the  somewhat  complicated  method  of  preparation  we  must 
refer  to  the  original  work  of  Hopkins  and  Cole. 

Skatosine,  CiiiHieNjOo,  is  a  base  first  obtained  by  Baum  in  the  pancreas  auto- 
digestion  and  later  studied  by  Swain.  It  develops  an  indol-  or  skatol-like  odor 
on  fusing  with  potassium  hydrate.  Langstein  ^  obtained  a  substance,  which  is 
perhaps  identical  with  skatosine,  in  the  very  lengthy  peptic  digestion  of  blood 
proteid. 

The  putrefactive  products  of  the  proteins  will  be  in  part  treated  in 
Chapter  IX  (intestinal  putrefaction)  and  in  part  in  Chapter  XV  (putre- 
factive products  in  the  urine). 

'  Journ.  of  Physiol.,  29;    see  also  EUinger  and  Gentzen,  Hofnaeister's  Beitrage,  4. 

^  Baum,  Hofmeister's  Beitrage,  3;    Swain,  ibid.;    Langstein,  see  Hofmeister,  t'ber 

Bau  und  Gruppierung  der  Eiweisskorper,  in  Ergebnisse  der  Physiologie,  I,  Abt,  1,  1902, 


CHAPTER  III. 
THE  CARBOHYDRATES 

We  designate  by  this  name  bodies  which  are  especially  abundant 
in  the  plant  kingdom.  As  the  protein  bodies  form  the  chief  portion  of 
the  solids  in  animal  tissues,  so  the  carbohydrates  form  the  chief  portion 
of  the  dry  substance  of  the  plant  structure.  They  occur  in  the  animal 
kingdom  only  in  proportionately  small  quantities,  either  free  or  in  com- 
binations with  more  complex  molecules,  forming  compound  proteids. 
Carbohydrates  are  of  extraordinarily  great  importance  as  food  for  both 
man  and  animals. 

The  carbohydrates  contain  only  carbon,  hydrogen,  and  oxygen.  The 
last  two  elements  occur,  as  a  rule,  in  the  same  proportion  as  they  do  in 
water,  namely,  2:1,  and  this  is  the  reason  why  the  name  carbohydrates 
has  been  given  to  them.  This  name  is  not  quite  pertinent,  if  strictly  con- 
sidered; because  we  not  only  have  bodies,  such  as  acetic  acid  and  lactic 
acid,  which  are  not  carbohydrates  and  still  have  their  oxygen  and  hydro- 
gen in  the  same  proportion  as  in  w^ater,  but  we  also  have  a  sugar  (rham- 
nose,  C6H12O5)  which  has  these  two  elements  in  another  proportion.  At 
one  time  it  was  thought  possible  to  characterize  as  carbohydrates  those 
bodies  which  contained  6  atoms  of  carbon,  or  a  multiple,  in  the  molecule, 
but  this  is  not  considered  tenable  at  the  present  time.  We  have  true  car- 
bohydrates containing  less  than  6,  and  also  those  containing  7,  8,  and  9 
carbon  atoms  in  the  molecule.  The  carbohydrates  have  no  properties  or 
characteristics  in  general  which  differentiate  them  from  other  bodies; 
on  the  contrary,  the  various  carbohydrates  are  in  many  cases  very  different 
in  their  external  properties.  Under  these  circumstances  it  is  very  difficult 
to  give  a  positive  definition  for  the  carbohydrates. 

From  a  chemical  standpoint  we  can  say  that  all  carbohydrates  are 
aldehyde  or  ketone  derivatives  of  polyhydric  alcohols.  The  simplest 
carbohydrates,  the  simple  sugars  or  monosaccharides,  are  either  aldehyde 
or  ketone  derivatives  of  such  alcohols,  and  the  more  complex  carbohydrates 
seem  to  be  derived  from  these  by  the  formation  of  anhydrides.  It  is  a 
fact  that  the  more  complex  carbohydrates  yield  two  or  even  more  molecules 
of  the  simple  sugars  when  made  to  undergo  hydrolytic  splitting. 

104 


MONOSACCHARIDES.  105 

The  carbohydrates  are  generally  divided  into  three  chief  groups,  namely, 
monosaccharides,  disacchandes ,  and  polysaccharides. 

Our  knowledge  of  the  carbohydrates  and  their  structural  relationships 
has  been  verj^  much  extended  by  the  pioneering  investigations  of  Kiliani  ^ 
and  especially  those  of  E.  Fischer.^ 

As  the  carbohydrates  occur  chiefly  in  the  plant  kingdom  it  is  naturally 
not  the  place  here  to  give  a  complete  discussion  of  the  numerous  carbo- 
hydrates known  up  to  the  present  time.  According  to  the  plan  of  this 
work  it  is  only  possible  to  give  a  short  re\'iew  of  those  carbohydrates  which 
occur  in  the  animal  kingdom  or  are  of  special  importance  as  food  for  man 
and  animals. 

Monosaccharides. 

All  varieties  of  sugars,  the  monosaccharides  as  well  as  disaccharides, 
are  characterized  by  the  termination  "ose,"  to  which  a  root  is  added  signi- 
fving  their  origin  or  other  relations.  According  to  the  number  of  carbon 
atoms,  or  more  correctly  oxygen  atoms,  contained  in  the  molecule  the 
monosaccharides  are  divided  into  trioses,  tetroses,  pentoses,  hexoses,  heptoses, 
and  so  on. 

All  monosaccharides  are  either  aldehydes  or  ketones  of  polyhydric 
alcohols.  The  former  are  termed  aldoses  and  the  latter  kefoses.  Ordinary 
dextrose  is  an  aldose,  while  ordinary  fruit  sugar  (levulose)  is  a  ketose.  The 
difference  may  be  shown  by  the  structural  formulse  of  these  two  varieties 
of  sugar: 

Dext  rose  =  CH2  (OH)  .CH  (OH)  .CH  (OH)  .CH  (OH)  .CH  (OH)  .CHO ; 
Levulose  =  CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(OH). 

A  difference  is  also  observed  on  oxidation.  The  aldoses  can  be  con- 
verted into  oxyacids  having  the  same  quantity  of  carbon,  while  the  ketoses 
yield  acids  ha\dng  less  carbon.  On  mild  oxidation  the  aldoses  yield  mono- 
basic oxyacids  and  dibasic  acids  on  more  energetic  oxidation.  Thus  ordi- 
nary dextrose  yields  gluconic  acid  in  the  first  case  and  saccharic  acid  in 
the  second. 

Gluconic  acid  =CH2(OH).[CH(OH)]4.COOH; 
Saccharic  acid  =  Co6H.[CH(OH)]4.COOH. 

The  monobasic  oxyacids  are  of  the  greatest  importance  in  the  artificial  forma- 
tion of  the  monosaccharides.     These  acids,  as  lactones,  can  be  converted  into 

'  Ber.  d.  deutsch,  chem.  Gesellsch.    18,  19,  and  20. 

^  See  E.  Fischer's  lecture,  Synthesen  in  der  Zuckergruppe,  Ber.  d.  deutsch.  chem. 
Gesellsch.,  23,  2114.  Excellent  works  on  carbohydrates  are  Tollens'  Kurzes  Hand- 
buch  der  Kohlehydrate,  Breslau,  2,  1895,  and  1,  2.  Auflage,  1898,  which  gives  a 
complete  review  of  the  literature,  and  E.  O.  v.  Lippmann,  Die  Chemie  der  Zucker- 
arten,  Braunschweig,  1904. 


106  THE  CARBOHYDRATES. 

their  respective  aldehydes  (corresponding  to  the  sugars)  by  the  action  of  nascent 
hydrogen.  On  the  other  hand,  they  may  be  transformed  into  stereoisomeric 
acids  on  heating  with  quinoline,  pyridine,  etc.,  and  the  stereoisomeric  sugars  may 
be  obtained  from  these  by  reduction. 

Xumerous  isomers  occur  among  the  monosaccharides,  and  especially  in 
the  hexose  group.  In  certain  cases,  as  for  instance  in  glucose  and  levulose, 
we  are  dealing  with  a  different  constitution  (aldoses  and  ketoses),  but  in 
most  cases  we  have  stereoisomerism  due  to  the  presence  of  asymmetric 
carbon  atoms. 

The  monosaccharides  are  converted  into  the  corresponding  polyhydric 
alcohols  by  nascent  hydrogen.  Thus  arabixose,  which  is  a  pentose, 
C5H10O5,  is  transformed  into  the  pentatomic  alcohol,  arabite,  C5H12O5. 
The  three  hexoses,  dextrose,  levulose,  and  galactose,  C6H12O6,  are 
transformed  into  the  corresponding  three  hexites,  sorbite,  mannite,  and 
dulcite,  C6Hi406-  In  these  reductions  a  second  isomeric  alcohol  is  also 
obtained;  in  the  reduction  of  levulose  we  obtain  besides  mannite  also 
sorbite.  Inversely,  the  corresponding  sugars  may  be  prepared  from 
polyhydric  alcohols  by  careful  oxidation. 

Like  the  ordinary  aldehydes  and  ketones,  the  sugars  may  be  made  to 
take  up  hydrocyanic  acid.  Cyanhydrins  are  thus  formed.  These  addition 
products  are  of  special  interest  in  that  they  make  possible  the  artificial  prepara- 
tion of  sugars  rich  in  carbon  from  sugars  poor  in  carbon. 

As  an  example,  if  we  start  from  dextrose  we  obtain  glucocyanhydrin  on  the 
addition  of  hydrocyanic  acid : 

CH2.(OH).[CH(OH)]4.COH+HCN=CH2(OH).[CH(OH)],.CH(OH).CX. 

On  the  saponification  of  glucocyanhydrin  the  corresponding  oxyacid  is  formed: 

CH2(OH).[CH(OH)],.CH(OH).CN+2H,0 

=  CH2(OH).[CH(OH)],.CH(OH).COOH  +  NH3. 

By  the  action  of  nascent  hydrogen  on  the  lactone  of  this  acid  we  obtain  gluco- 
heptose,  C7H14O7. 

The  monosaccharides  give  the  corresponding  oximes  with  hvdroxylamine ; 
thus  glucose  yields  glucosoxime,  CH,(OH).[CH(OH)],.CH:N.OH.*^  These  com- 
pounds are  of  importance  on  account  of  the  fact,  as  found  by  Wohl,'  that 
they  are  the  starting-point  in  the  formation  of  one  class  of  sugars  from  another 
class,  namely,  the  preparation  of  sugars  poor  in  carbon  from  those  rich  in  carbon. 

The  monosaccharides  are  strong  reducing  bodies,  similar  to  the  alde- 
hydes. They  reduce  metallic  silver  from  ammoniacal  silver  solutions,  and 
also  several  metallic  oxides,  such  as  copper,  bismuth,  and  mercury  oxides, 
on  warming  their  alkaline  solutions.  This  property  is  of  the  greatest 
importance  in  their  detection  and  quantitative  estimation. 

With  phenylhydrazine  or  substituted  phenylhydrazines,  the  sugars  first 
yield  hydrazones  with  the  elimination  of  water,  and  then  on  the  further 
action  of  hydrazine  on  warming  in  an  acetic-acid  solution  we  obtain  osazones. 


»  Ber.  d.  d.  chem.  Gesellsch.,  2G.  p.  730. 


MONOSACCHARIDES.  107 

The  reaction  takes  place  as  follows: 

(a)  CH,(0H).[CH(0H)]3.CH(0H).CH0  +  HoN.XH.CH^ 

=  CH,(0H).[CH(bH)]3.CH(0H)CH:N.NH.C,H5  +  H,0. 

Phenylglucosehydrazone. 

(b)  CH,(0H)[CH(0H)]3.CH(0H).CH:N.NH.C,H,+H,N.XH.C,H5 

=  CH,{0H).[CH(0H)]3.C.CH:N.NH.C6H, 

N.NH.C,H5  +  H,0  +  H,. 

Phenylglucosazone. 

The  hydrogen  is  not  evolved,  but  acts  on  a  second  molecule  of  phenylhy- 
drazine  and  splits  it  into  aniline  and  ammonia: 

H,N.NHAH5  +  H,  =  H,N.C6H,  +  NH,. 

The  osazones  are  generally  yellow  crystalline  compounds  which  differ 
from  each  other  in  melting-point,  solubility,  and  optical  properties,  and 
hence  have  been  of  great  importance  in  the  characterization  of  certain 
sugars.  They  have  also  become  of  extraordinarily  great  interest  in  the 
study  of  the  carbohydrates  for  other  reasons.  Thus  they  are  a  ver}^  good 
means  of  precipitating  sugars  from  solution  in  which  they  occur  mixed 
with  other  bodies,  and  they  are  of  the  greatest  importance  in  the  artificial 
preparation  of  sugars.  On  cleavage,  by  the  brief  action  of  gentle  heat 
and  fuming  hydrochloric  acid  (for  disaccharides  still  better  with  benzalde- 
hyde)  ^  the  osazones  yield  so-called  osones,  which  on  reduction  yield  aldoses 
or  more  often  ketoses. 

We  can  also  pass  from  the  osazones  to  the  corresponding  sugars 
(ketoses)  in  other  ways,  namely,  by  direct  reduction  of  the  osazones  \Aith 
acetic  acid  and  zinc  dust.  The  corresponding  osamine  is  first  formed 
(from  phenylglucosazone  we  obtain  isoglucosamine),  which  on  treatment 
with  nitrous  acid  yields  the  sugar  (in  this  case  le\ailose). 

The  sugars  can  be  prepared  from  the  hydrazones  by  decomposition 
with  benzaldehyde  (Herzfeld)  or  with  formaldehyde  (Ruff  and  Ollen- 
dorff 2).  This  latter  method  is  especially  applicable  if  substituted  hydra- 
zines, especially  benzylphenylhydrazine,  are  used. 

With  ammonia  the  glucoses  may  form  compounds  which  have  been 
considered  as  osamines  by  Lobry  de  Bruyn,  but  to  differentiate  them  from 
the  true  osamines  have  been  called  osimines  by  E.  Fischer.^  The  corre- 
sponding osaminic  acid  can  be  obtained  from  such  an  osimine  by  the  action 
of  ammonia  and  hydrocyanic  acid,  and  from  the  hydrochloric-acid  lactone 
of  this  acid  the  osamine  is  obtained  by  reduction  with  sodium  amalgam. 
In  this  manner  E.  Fischer  and  Leuchs  artificially  prepared  c?-glucosa- 
mine,  which  occurs  in  the  animal  kingdom  and  is  an  isomer  of  the  above- 


'  E.  Fischer  and  Arinstronsr,  Ber..  d.  d.  chem.  Gesellsch.,  35. 
2  Herzfeld,   ibid.,  28;     Ruff  and   OllendorlT,   ibid.,   32. 
'Lobry  de  Bruyn,  ibid.,  2S;    E.  Fischer,  ibid.,  35. 


108  THE  CARBOHYDRATES. 

mentioned  isoglucosamine,  by  starting  from  d-aral)inose,  then  obtaining 
rf-aral^inosimine,  then  d-gkicosaminic  acid,  and  finally  the  glucosamine 
from  the  lactone  of  this  acid.  They  ^  also  have  prepared  Z-glucosamine 
from  Z-arabinose  in  a  similar  manner. 

Knoop  and  Windaus  -  have  obtained  large  amounts  of  methylimidazol, 
CH3 

C — NHv         ,  from  glucose  by  the  action  of  ammonium-zinc  hydroxide 

I!  >H 

CH— N^ 

at  ordinary  temperatures.  A  genetic  relationship  of  the  carbohydrates  to 
histidine  and  the  purine  bodies  is  thus  made  probable  by  the  imidazol  for- 
mation. 

By  the  action  of  hydrochloric  acid  upon  alcoholic  sugar  solutions  E. 
Fischer  and  his  pupils  have  obtained  ether-like  compounds  which  have 
been  called  gincosides.  Compounds  with  aromatic  groups  similar  to  the 
glucosides  occur  widely  distributed  in  the  vegetable  kingdom.  The  more 
complex  carbohydrates  may  be  considered,  according  to  Fischer,  as 
glucosides  of  the  sugars.  Thus  maltose,  for  example,  is  the  glucoside 
and  lactose  the  galactoside  of  dextrose. 

By  the  action  of  alkalies,  even  in  small  amounts,  as  also  of  alkaline 
earths  and  lead  hydroxide,  a  reciprocal  transformation  of  the  sugars,  such 
as  dextrose,  levulose,  and  mannose,  may  take  place  (Lobry  de  Bruyn  and 
Alberda  van  Ekenstein^). 

Four  other  sugars,  among  them  two  ketoses,  are  produced  by  the  action  of 
potash  or  soda  on  each  of  the  three  sugars,  dextrose,  levulose,  and  galactose. 
For  example,  from  dextrose  two  ketoses,  levulose  and  pseudolevulose,  are  pro- 
duced, also  mannose  and  a  non-fermentable  sugar,  glutose.  From  galactose 
are  formed  talose  and  galtose,  besides  two  ketoses,  tagatose  and  pseudotagatose. 

The  transformation  of  the  different  varieties  of  sugar  into  each  other 
also  occurs  in  the  animal  body.  Neuberg  and  Mayer'*  have  shown  by 
experiments  on  rabbits  the  partial  transformation  of  various  mannoses  into 
the  corresponding  glucoses. 

The  monosaccharides  are  colorless  and  odorless  bodies,  neutral  in  re- 
action, with  a  sweet  taste,  readily  soluble  in  water,  generally  soluble  with 
difficulty  in  absolute  alcohol,  and  insoluble  in  ether.  Some  of  them  crys- 
tallize well  in  the  pure  state.  They  are  optically  active,  some  levorotatory 
and  others  dextrorotatory;    but  there  are  also  optically  inactive  modi- 

'  Ber.  d.  d.  chem.  Gesellsch.,  35,  p.  3787,  and  36,  p.  24. 
^  Ibid.,  38,  and  Hofmeister's  Boitrage,  6. 

'Ber.  d.  d.  chem.  Gesellsch.,  2S,  3078;    Bull.  see.  chim.  de  Paris  (3),  15;    Chem. 
Centralbl.,  1896,  2,  and  1897,  2. 
•"  Zoitschr.  f.  physiol.  Chem.,  37. 


MONOSACCHARIDES.  109 

fications  (racemic),  which  are  formed  from  two  opti'^-ally  opposed  compo- 
nents. 

We  designate  the  optical  activity  of  the  carbohydrates  with  the  letter 
I-  for  levogyrate,  d-  for  dextrogyrate,  and  i-  for  inactive.  These  are  only 
partly  indicative.  Thus  dextrorotatory  glucose  is  designated  cZ-glucose, 
levorotatory  /-glucose,  and  the  inactive  ^'-glucose.  Emil  Fischer  has  used 
these  signs  in  another  sense.  He  designates  by  these  signs  the  mutual 
relationship  of  the  various  kinds  of  sugars  instead  of  their  optical  activity. 
For  example,  he  does  not  designate  the  levorotatory  levulose  Z-le^allose, 
but  rf-le^1llose,  showing  its  close  relation  to  dextrorotatory  d-glucose. 
This  designation  is  generally  accepted,  and  the  above-mentioned  signs  only 
show  the  optical  properties  in  certain  cases. 

Specific  rotation  means  the  rotation  in  degrees  produced  by  1  gn.  substance 
dissolved  in  1  c.c.  liquid  placed  in  a  tube  1  dcm.  long.  The  reading  is  ordinarily 
made  at  20°  C.  and  with  the  monochromatic  sodium  light.  The  specific  rotation 
with  this  light  is  represented  by  (a)D,  and  is  expressed  by  the  following  formula: 

(a)D=  it — -,  in  which  a  represents  the  reading  of  degrees,  1  the  length  of  the 

tube  in  decimetres,  and  p  the  weight  of  substance  in  1  c.c.  of  the  liquid.     In- 
versely the  per  cent  P  of  substance  can  be  calculated,  when  the  specific  rotation 

is  known,  by  the  formula  P  =  — — ,  in  which  s  represents  the  known  specific 

s.l 

rotation. 

A  freshly  prepared  sugar  solution  often  shows  a  different  rotation  from  one 

Avhich  has  been  allowed   to    stand   for   some    time.      If  the  rotation  gradually 

diminishes,  this  is  called  birotation,  while  a  gradual  increase  in  the  rotation  is 

called  half -rotation. 

]\Ian3'  monosaccharides,  but  not  all,  ferment  with  3'east,  and  it  has  been 
shown  that  only  (hose  varieties  of  sugar  containing  3,  6,  or  9  atoms  of 
carbon  in  the  molecule  are  fermentable  with  yeast.  We  must  state,  how- 
ever, that  the  power  of  fermentation  with  pure  yeast  has  been  shown  only 
for  the  hexose  group,  and  in  fact  all  of  the  hexoses  do  not  ferment.  The 
restriction  of  fermentation  to  only  certain  monosaccharides  is,  accord- 
ing to  E.  Fischer,  like  the  action  of  the  inverting  enzymes  upon  disac- 
charides  and  glucosides,  dependent  upon  the  stereometric  configuration  of 
the  sugars  (see  Chapter  I).  This  difference  in  configuration  is  important 
not  only  for  the  action  of  lower  living  organisms  upon  the  sugars,  but 
also  upon  the  behavior  of  the  sugars  within  more  highly  developed  organ- 
isms. Thus  the  investigations  of  Neuberg  and  Wohlgemuth  1  upon  ara- 
binose  and  of  Neuberg  and  Mayer  2  on  mannoscs  have  shown  that  in 
rabbits  the  /-arabinose  and  the  tZ-mannose  are  much  better  utilized  than  d- 
and  /-arabinose  or  /-  and  /-mannose,  and  they  have  also  shown  that  the 
lower   organisms   have   the   tendency   toward   decomposing   inactive   sub- 

*  Zeitschr.  f.  physiol.  Chem.,  35.  'Ibid.,  37. 


110  THE  CARBOHYDRATES 

stances  into  their  optically  active  components  to  a  much  higher  degree 
than  the  higher  organisms. 

By  the  action  of  lower  organisms  of  various  kinds  the  sugars  may  be 
made  to  undergo  fermentations  of  different  kinds,  such  as  lactic-acid  and 
butyric-acid  fermentation  and  mucilaginous  fermentation. 

The  simple  varieties  of  sugar  occur  in  part  in  nature  as  such,  already 
formed,  which  is  the  case  with  both  of  the  very  important  sugars,  dextrose 
and  levulose.  They  also  occur  in  great  abundance  in  nature  as  more 
complex  carbohydrates  (di-  and  polysaccharides);  also  as  ester-like  com- 
binations with  different  substances,  as  so-called  glucosides. 

Among  the  groups  of  monosaccharides  known  at  the  present  time,  those 
containing  less  than  five  and  more  than  six  carbon  atoms  in  the  molecule 
have  no  great  imjjortance  in  biochemistry,  although  the}^  are  of  high  scien- 
tific interest.  Of  the  other  tw^o  groups  the  hexoses  are  of  the  greatest 
importance,  hence  in  the  past  only  those  carbohydrates  with  six  carbon 
atoms  w^ere  considered  as  tnie  carbohydrates.  As  the  pentoses  have  been 
the  subject  of  numerous  biochemical  investigations  of  late,  they  will  also 
be  discussed  in  brief. 

Pentoses  (C5H10O5). 

As  a  rule  the  pentoses  do  not  occur  as  such  in  nature,  but  are  formed  in 
the  hydrolytic  splitting  of  several  more  complex  carbohydrates,  the  so- 
called  pentosanes,  especially  on  boiling  gums  with  dilute  mineral  acids. 
The  pentosanes  exist  very  widely  distributed  in  the  plant  kingdom  and  aro 
especially  of  great  importance  in  the  building  up  of  certain  plant  con- 
stituents. The  pentoses  were  first  found  b}'  Salkowski  and  Jastrowitz 
in  the  animal  kingdom  in  the  urine  of  a  person  addicted  to  the  morphine 
habit,  and  later  by  Salkowski  and  others  in  normal  human  urine.  Small 
quantities  of  pentoses  have  been  detected  by  KiJLZ  and  Vogel  ^  in  the 
urine  of  diabetics,  as  also  in  dogs  with  pancreas  diabetes  or  phlorhizin 
diabetes.  Pentose  has  also  been  found  by  Hammarstex  amongst  the 
cleavage  products  of  a  nucleoproteid  obtained  from  the  pancreas,  and 
saems  also,  according  to  the  observations  of  Blumenthal,  to  be  a  constit- 
uent of  nucleoproteids  of  various  organs,  such  as  the  thymus,  thyroid, 
brain,  spleen,  and  liver.  In  regard  to  the  quantity  of  pentoses  found  in 
the  various  organs,  we  must  refer  to  the  works  of  Gruxd  and  of  Bexdix 
and  Ebsteix.2 


'Salkowski  and  Jastrowitz  Centralbl  i,  d.  med.  Wissensch.  1892.  337  and  593; 
Salkowski,  Berl.  klin.  Wochenschr.,  1895,  Bial  Zeitschr.  f.  klin.  Med..  39;  Bial  and 
Blumenthal,  Deutsch.  med.  Wochenschr.,  1901,  No.  2;  Kiilz  and  Vogel,  Zeitschr.  f. 
Biologie,  32. 

^  Hammarsten  Zeitschr  f.  physiol.  Chem.,  19;  also  Salkowski,  Berl.  klin.  Wochen- 
schr., 1895;  Blumenthal  Zeitschr.  f.  klin.  Med.,  34;  Grund,  Zeitschr.  f.  physiol.  Chem.. 
3.';    Bendix  and  Ebstein    Zeitschr.  f.  allgemein.  Physiol.,  2. 


PENTOSES.  11] 

The  pentosanes  (Stone,  Slowtzoff)  as  well  as  the  pentoses  are  of  the 
greatest  importance  as  foods  for  herbivorous  animals.  In  regard  to  the 
value  of  the  pentoses,  the  researches  of  Salkowski,  Cremer,  Neuberg, 
and  Wohlgemuth  ^  upon  rabbits  and  hens  show  that  these  animals  car- 
utilize  the  pentoses.  The  question  whether  the  pentoses  are  active  as 
glycogen-formers  is  still  an  open  one  (see  Chapter  VIII).  The  pentoses 
seem  to  be  absorbed  by  human  beings  and  in  part  utilized,  but  they  pass 
in  part  into  the  urine  even  when  small  quantities  are  taken .^ 

The  natural  pentoses  are  reducing  aldoses,  and  as  a  rule  do  not  belong 
to  the  sugars  fermentable  with  yeast.  Still,  the  observations  of  Salkowski. 
Bendix,  Schoxe  and  Tollens  seem  to  indicate  that  the  pentoses  are 
fermentable.^  They  are  readily  decomposed  by  putrefaction  bacteria. 
With  phenylhydrazine  and  acetic  acid  they  give  crystalline  osazones  which 
are  soluble  in  hot  water  and  whose  melting-points  and  optical  behavior  are 
important  for  the  detection  of  the  pentoses.  On  heating  with  hydrochloric 
acid  they  yield  furfurol,  but  no  le\ailinic  acid.  The  furfurol  passing  over 
on  distilling  with  hydrochloric  acid  can  be  detected  by  the  aid  of  aniline- 
acetate  paper,  which  is  colored  beautifully  red  by  furfurol.  In  the  quan- 
titative estimation  we  can  use  the  method  suggested  by  Tollex.s,  which 
consists  of  converting  the  furfurol  in  the  distillate  into  phloroglucide  by 
means  of  phloroglucin  and  weighing  (see  Tollens  and  Krober,  Grund, 
Bendix  and  Ebsteix).^  These  methods  are  still  not  quite  accurate,  to 
say  nothing  of  the  fact  that  glucuronic-acid  compounds  also  yield  furfurol 
under  the  same  conditions.  The  two  following  pentose  reactions,  as  sug- 
gested by  Tollexs,  are  especially  applicable. 

The  orcin-hydrochloric  acid  test.  Mix  with  the  solution  or  the  sub- 
stance introduced  into  water  an  equal  volume  of  concentrated  hydrochloric 
acid,  add  some  orcin  in  substance,  and  heat.  In  the  presence  of  pentoses 
the  solution  becomes  reddish  blue,  then  bluish  green,  and  on  spectroscopic 
examination  an  absorption-band  is  observed  between  C  and  D.  If  i: 
is  cooled  and  shaken  with  amyl  alcohol,  a  bluish-green  solution  which 
shows  the  same  band  is  obtained. 

The  phloroglucin-hydrochloric  acid  test.  This  test  is  performed  in 
the  same  manner  as  the  above,  using  phloroglucin  instead  of  orcin.     The 


'  S'one,  Amer.  Chem.  Journ.,  14;  Slowtzoff,  Zeitschr.  f.  physiol.  t^Jhem.,  34;  Sal- 
kowski, 1.  c,  Centralbl.;  Cremer,  Zeitschr.  f.  Biologie,  29  and  42;  Neuberg  and  Wohl- 
gemuth, Zeitschr.  f.  physiol.  Cliem.,  3.5. 

^  See  Ebstein,  Virchow's  Arch.,  129;  Tollens,  Ber.  d.  deutsch.  chem.  Gesellsch..  29, 
1208;  Cremer,  1.  c;  Lindemann  and  May,  Deutsch.  Arch.  f.  khn.  Med.,  56;  Sal- 
kowski, Zeitschr.  f.  physiol.  Chem..  30. 

'Salkowski.  Zeitschr.  f.  physiol.  Chem.,  30;.  Bendix,  see  Chem.  Centralbl.,  1900,  1; 
Schone  and  Tollens.  ibid.,  1901,  1. 

^Bendix  and   Ebstein,   1.   c,   which   contains   the   literature. 


112  THE  CARBOHYDRATES. 

solution  becomes  cherry-red  on  heating  and  then  becomes  cloudy  and  hence 
a  shaking  out  with  amyl  alcohol  is  very  necessary.  The  red  amyl-alcohol 
solution  shows  an  absorption-band  between  D  and  E.  The  orcin  test  is 
better  for  several  reasons  than  the  phloroglucin  test  (Salkowski  and 
Neuberg  ^).  In  regard  to  the  use  of  these  tests  in  urine  examination 
see  Chapter  XV. 

Many  modifications  of  these  tests  have  been  suggested.  Brat  ^  performs 
the  orcin  reaction  by  the  addition  of  NaCl  and  heating  to  only  90-95°.  Bial  ^ 
uses  a  hydrochloric  acid  containing  ferric  chloride  for  the  orcin  test  and  claims 
to  get  a  greater  delicacy.  On  too  strong  and  too  long  heating  (1^-2  minutes), 
when  using  this  modification,  a  confusion  with  sugars  of  the  six  carbon  series  may 
occur  (Bial,  van  Leersum).*  According  to  R.  Adler  and  O.  Adler  the  phlo- 
roglucin and  orcin  tests  can  be  made  with  glacial  acetic  acid  and  a  few  drops 
hydrochloric  acid  instead  of  with  the  hydrochloric  acid  alone.  These  investigators 
also  use  a  mixture  of  equal  volumes  of  aniline  and  glacial  a  etic  acid  as  a  reagent 
for  pentoses.  On  the  addition  of  a  little  pentose  to  the  boiling  mixture  a  beautiful 
red  color  of  furfurol-aniline  acetate  is  obtained.  A.  Neumann  ^  performs  the 
orcin  test  with  glacial  acetic  acid  and  adds  concentrated  sulphuric  acid  drop 
by  drop.  On  following  the  exact  instructions  not  only  do  the  pentoses  give 
this  reaction,  but  also  glucuronic  acid,  dextrose,  and  levulose  give  characteristic 
colored  solutions  with  special  absorption-bands  which  can  be  made  use  of  in 
identifying  the  various  sugars. 

In  performing  the  above  two  tests  for  pentose  it  must  be  borne  in  mind 
that  glucuronic  acid  gives  the  same  reactions  and  also  that  the  colors 
alone  are  not  sufficient.  The  spectroscopic  examination  must  therefore 
never  be  omitted.  Both  tests  are  to  be  considered  as  tests  of  detection 
rather  than  definite  pentose  reactions,  and  therefore  for  a  positive  detection 
of  pentoses  we  must  prepare  also  the  osazones  or  other  compounds. 

Arabinoses.  The  pentose  isolated  by  Neuberg®  from  human  urine 
is  2-arabinose.  It  can  be  isolated  from  the  urine  as  the  diphenylhydrazone, 
from  w^hich  the  arabinose  can  be  separated  by  splitting  with  formaldehyde. 
The  2-arabinose  is  crystalline,  has  a  sweetish  taste,  is  optically  inactive, 
and  melts  at  163-164°  C.  Its  di])henylhydrazone,  which,  according  to 
Neuberg  and  Wohlgemuth,'^  can  be  used  in  its  quantitative  estimation, 
melts  at  206°  C,  is  insoluble  in  cold  water  and  alcohol,  but  readily  soluble 
in  pyridine.     The  osazone  melts  at  166-168°  C. 

The  dextrorotatory  Z-arabinose  is  obtained  by  boiling  gum  arable  or 
cherry  gum  with  dilute  sulphuric  acid.  The  c?-arabinose  is  prepared  syn- 
thetically.   The  diphenylhydrazone  of  Z-arabinose  has  according  to  Neuberg 

'  Salkowski,  Zeitschr.   f.  physiol.   Cbem.,  27;    Neuberg,  ihid.,  31. 

^Zeitschr.  f.  klin.  Med.,  47. 

^Deutsch.  med.  Wochenschr.,  1902  and  1903,  and  Zeitschr.  f.  klin.  Med.,  50. 

*  Bial,  Zeitschr.  f.  klin.  Med.,  50;    van  Leersum,  Hofmeister's  Beitrage,  5. 

*  R.  and  O.  Adler,  Pfliiger's  Arch.,  106;  A.  Neumann,  Berl.  klin.  Wochenschr., 
1904. 

"  Ber.  d.  d.  chem.  Gesellsch.,  33. 

'  Zeitschr.  f.  physiol.  Chem.,  35.  , 


HEXOSES.  113 

a  melting-point  of  216-218°  C,  while  according  to  Tollens  and  ^Iauren- 
brecherI  i^g  melting-point  is  204-205°. 

Xyloses.  The  only  pentose  thus  far  isolated  from  the  animal  tissues 
is  /-xylose,  obtained  by  Neuberg  from  the  pancreas  proteins,  and  is 
identical  with  the  xylose  found  widely  distributed  in  the  plant  kingdom 
and  obtained  from  wood-gum  by  boiling  with  dilute  acids.  Xylose  is 
crystalline,  melts  at  153-154°  C,  dissolves  very  readily  in  water  but  ^^•ith 
difficulty  in  alcohol,  is  faintly  dextrorotatory,  (o)d=  +18.1°,  and  gives  a 
phenylosazohe  which  melts  at  159-160°  C,  and  according  to  Tollexs  and 
^MiJTHER^  a  diphenylhydrazone  which  melts  at  107-108°.  Xylose  can 
be  transformed  into  xylonic  acid,  CH20H(CHOH)3COOH,  by  bromine- 
water,  and  the  brucine  salt  of  this  acid  is,  according  to  Neuberg,  well  suited 
for  the  detection  and  isolation  of  xylose. 

Hexoses  (C6Hi206). 

The  most  important  and  best-known  simple  sugars  belong  to  this  group, 
and  most  of  the  other  bodies  which  have  been  considered  as  carbohydrates 
in  the  past  are  anhydrides  of  this  group.  Certain  hexoses,  such  as  dextrose 
and  levulose,  either  occur  in  nature  already  formed  or  are  produced  by  the 
hydrolytic  splitting  of  other  more  complicated  carbohydrates  or  glucosides. 
Others,  such  as  mannose  or  galactose,  are  formed  by  the  hydrolytic  cleavage 
of  other  natural  products;  while  some,  on  the  contrary,  such  as  gulose, 
talose,  and  others,  are  obtained  only  by  artificial  means. 

All  hexoses,  as  also  their  anhydrides,  yield  levulinic  acid,  C5H8O3. 
besides  formic  acid  and  humus  substances  on  boiling  with  dilute  mineral 
acids.  Some  of  the  hexoses  are  fermentable  \Wth  yeast,  while  the  artificially 
prepared  hexoses  are  not,  or  at  least  only  incompletely  and  with  great 
difficulty. 

Some  hexoses  are  aldoses,  while  others  are  ketoses.  Belonging  to  the 
first  group  we  have  mannose,  dextrose,  gulose,  galactose,  and  talose, 
and  to  the  other  levulose,  and  possibly  also  sorbinose.  We  differen- 
tiate also  between  the  d,  I,  and  i  modifications;  for  instance,  d-,  1-,  and 
t-dextrose;  hence  the  number  of  isomers  is  very  great. 

The  most  important  syntheses  of  the  carbohydrates  have  been  made  by 
E.  Fischer  and  his  pupils  chiefiy  within  the  members  of  the  hexose  group. 
A  short  summar}^  of  the  syntheses  of  hexoses  is  given  below. 

The  first  artificial  preparation  of  a  sugar  was  made  by  Butlerow.  On 
treating  trioxymethylene,  a  polymer  of  formaldehyde,  with  lime-water  he  ob- 

'  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  35^  and  Ber.  d.  d.  chem,  Gesellsch.,  33; 
ToUens  and  Maurenbrecher,  ibid.,  38. 

'Neuberg,  Ber.  d.  d.  chem.  Gesellsch.,  35;    Tollens  and  Muther,  ibid.,  37. 


114  THE  CARBOHYDRATES. 

tained  a  faintly  sweetish  syrup  called  methylenitan.  Loew  '  later  obtained  a 
jnixture  of  several  sugars,  from  which  he  isolated  a  fermentable  sugar,  called 
methose,  by  condensation  of  formaldehyde  in  the  presence  of  bases.  The  most 
important  and  comprehensive  syntheses  of  sugar  have  been  performed  by  E. 
Fischer.^ 

The  starting-point  of  these  syntheses  is  a-acrose,  which  occurs  as  a  condensa- 
tion product  of  formaldehyde.  The  name  a-acrose  has  been  given  to  this  body 
because  it  is  obtained  from  acrolein  bromide  by  the  action  of  bases  (Fischer). 
It  is  also  obtained  admixed  with  [i-acrose  on  the  oxidation  of  glycerine  with 
bromine  in  the  presence  of  sodium  carbonate  and  treating  the  resulting  mixture 
with  alkali.  On  the  oxidation  with  bromine  a  mixture  of  glvcerine  aldehvde, 
CH^OH.CHCOHj.CHO,  and  dioxyacetone,  CH^COHj.CO.CH^OH,  is  obtained. 
These  two  bodies  may  be  considered  as  true  sugars,  nameh',  glyceroses  or  trioses. 
It  seems  as  if  a  condensation  to  hexoses  takes  place  on  treatment  with  alkalies. 

a-acrose  may  be  isolated  from  the  above  mixture  and  obtained  pure  by  first 
converting  it  into  its  osazone  and  then  retransforming  this  into  the  sugar. 
«-acrose  is  identical  with  ?'-levulose.  With  yeast  one  half,  the  levogyrate  d-levu- 
lose,  ferments,  while  the  dextrogyrate  Z-levulose  remains.  The  i-  and  /-levulose 
may  be  prepared  in  this  waj'. 

On  the  reduction  of  a-acrose  we  obtain  a-acrite,  which  is  identical  with  {-man- 
nite.  On  oxidation  of  ?'-mannite  we  obtain  i'-mannose,  from  which  only  /-man- 
nose  remains  on  fermentation.  On  further  oxidation  of  {-mannose  it  j^elds 
1-mannonic  acid.  The  two  active  mannonic  acids  may  be  separated  from  each 
other  by  the  fractional  crystallization  of  their  strychnine  or  morphine  salts.  The 
two  corresponding  mannoses  may  be  obtained  from  these  two  acids,  d-  and 
/-mannonic  acids,  by  reduction. 

d-Levulose  is  obtained  from  d-mannose  by  the  method  given  on  page  107,  using 
the  osazone  as  an  intermediate  step.  The  d-  and  /-mannonic  acids  are  |3artly 
converted  into  d-  and  /-gluconic  acids  on  heating  with  quinoline,  and  d-  or  /-glucose 
is  obtained  on  the  reduction  of  these  acids;  /-glucose  is  best  prepared  from 
/-arabinose  by  means  of  the  cyanhydrin  reaction,  using  /-gluconic  acid  as  the 
intermediate  step.  The  combination  of  /-  and  d-glueonic  aeids,  forming  {-glu- 
conic acid,  yields  ?-glucose  on  reduction. 

The  artificial  preparation  of  sugars  by  means  of  the  condensation  of  formalde- 
hyde has  received  special  interest  because,  according  to  Baeyer's  assimilation 
hypothesis,  in  plants  formaldehyde  is  first  formed  by  the  reduction  of  carbon 
dioxide,  and  the  sugars  are  produced  by  the  condensation  of  this  formaldehyde. 
BoKORNY  ■*  has  shown,  by  special  experiments  on  algse  Spirogyra,  that  formalde- 
hyde .sodium  sulphite  was  split  by  the  living  algse  cells.  The  formaldehyde  set 
free  is  immediately  condensed  to  carbohydrate  and  precipitated  as  starch. 

Among  the  hexoses  kno^^Tl  at  the  present  time  only  dextrose,  levulose 
and  galactose  are  really  of  physiological-chemical  interest;  therefore  the 
other  hexoses  will  be  only  incidentally  mentioned. 

Dextrose  (d-glucose) — glucose,  grape-sugar,  and  diabetic  sugar — 
occurs  abundantly  in  the  grape,  and  also,  often  accompanied  with  le^1dose 
(J-fructose),  in  honey,  sweet  fruits,  seeds,  roots,  etc.  It  occurs  in  the 
human  and  animal  intestinal  tract  during  digestion,  also  in  small  quantities 
in  the  blood  and  lymph,  and  as  traces  in  other  animal  fluids  and  tissues. 


'  Butlerow,  Ann.  d.  Chem.  u.  Pharm.,  120;   Compt.  rend.,  53;    O.  Loew   Jouin.  f. 
prakt.  Chem.  (N.  P.),  33,  and  Ber.  d.  deutsch.  chem.  Gesellsch.    20,  21,  22. 
^  Ber.  d.  d.  chem.  Gesellsch.,  21,  and   1.   c,  p.   105. 
*Biolog.  Centralbl.,  J2,  pp.  321  and  481. 


DEXTROSE.  115 

It  occurs  only  as  traces  in  urine  under  normal  conditions,  while  in  diabetes 
the  quantity  is  ^•er^'  large.  It  is  formed  in  the  hydrolytic  cleavage  of 
starch,  dextrin,  and  other  compound  carbohydrates,  as  also  m  the  splitting 
of  glucosides.  The  question  whether  dextrose  can  be  formed  in  the  bodj^ 
from  proteins  or  from  fats  is  disputed  and  will  be  discussed  in  a  following 
chapter  (VIU). 

Properties  of  Dextrose.  Dextrose  ciystallizes  sometimes  with  1  molecule 
of  water  of  crj'stallization  in  warty  masses  or  small  leaves  or  plates,  and 
sometimes  when  free  from  water  in  needles  or  prisms.  The  sugar  contain- 
ing water  of  cr\-stallization  melts  even  below  100°  C.  and  loses  its  water 
of  crystallization  at  110°  C.  The  anhydrous  sugar  melts  at  146°  C,  and 
is  converted  into  glucosan,  CeHioOs,  at  170°  C.  -uith  the  elimination  of 
water.  On  strongly  heating  it  is  converted  into  caramel  and  then  de- 
composes. 

Dextrose  is  readily  soluble  in  water.  This  solution,  which  is  not  as 
sweet  as  a  cane-sugar  solution  of  the  same  strength,  is  dextrogyrate  and 
shows  strong  birotation.  The  specific  rotation  is  dependent  upon  the 
concentration  of  the  solution,  as  it  increases  with  an  increase  in  the  con- 
centration. A  10  per  cent  solution  of  anhydrous  glucose  can  be  taken  as 
52.74°  at  20°  C.^  Dextrose  dissolves  sparingly  in  cold,  but  more  freely 
in  boiling  alcohol.  100  parts  alcohol  of  sp.  gr.  0.837  dissolves  1.95  parts 
anhydrous  dextrose  at  17.5°  C.  and  27.7  parts  at  the  boilmg  temperature 
(AxTHOx^).     Dextrose  is  insoluble  in  ether. 

If  an  alcoholic  caustic-potash  solution  is  added  to  an  alcoholic  solution 
of  dextrose,  an  amorphous  precipitate  of  insoluble  sugar-potash  compound 
is  formed.  On  warming  this  compound  it  decomposes  easih'  with  the 
formation  of  a  yellow  or  bro'WTiish  color,  which  is  the  basis  of  !\Joore's 
test.     Dextrose  forms  also  compounds  with  lime  and  bars'ta. 

Moore's  Test.  If  a  dextrose  solution  is  treated  with  about  one  quarter 
of  its  volume  of  caustic  potash  or  soda  and  warmed,  the  solution  becomes 
first  yellow,  then  orange,  yellowish  brown,  and  lastly  dark  brouTi.  It  has 
at  the  same  time  a  faint  odor  of  caramel,  and  this  odor  is  more  pronounced 
on  acidification.-^ 

Dextrose  forms  several  cr\-stallizable  combinations  vdth.  NaCl,  of  which 
the  easiest  to  obtain  is  (C6Hi206)2-NaCH-H20,  which  forms  large  colorless 
six-sided  double  pyramids  or  rhomboids  with  13.52  per  cent  NaCl. 

Dextrose  in  neutral  or  verj^  faintly  acid  (organic  acid)  solution  under- 
goes alcoholic  fermentation  ^ith  beer-yeast:  C6Hi206  =  2C2H5.0H  +  2C0o. 
Besides  the  alcohol  and  carljon  dioxide  there  are  formed,  especiallv  at 

*  For  further  information  see  Tollens,  Handbuch  der  Kohlehydrate,  2.  Aufi.    44. 
^  Cited  from  ToUens'  Handbuch. 

^  In  regard  to  the  products  formed  in  this  reaction,  see  Framm,  Pfliiger's  Arch.,  64- 
and  especially  Gaud,  Compt.  rend.,  119. 


116  THE  CARBOHYDRATES. 

higher  temperatures,  small  quantities  of  homologous  alcohols  (amyl 
alcohol),  glycerine,  and  succinic  acid.  In  the  presence  of  acid  milk  or 
cheese  the  dextrose  undergoes  lactic-acid  fermentation,  especially  in  the 
presence  of  a  base  such  as  ZnO  or  CaCOs.  The  lactic  acid  may  then 
further  undergo  butyric-acid  fermentation:  2C3H603  =  C4H802  +  2C02  +  4H. 

Dextrose  reduces  several  metallic  oxides,  such  as  copper  oxide,  bismuth 
oxide  and  mercuric  oxide,  in  alkaline  solutions,  and  the  most  important 
reactions  for  sugar  are  based  on  this  fact. 

Trommer's  test  is  based  on  the  property  that  dextrose  possesses  of  re- 
ducing cupric  hj^drate  in  alkaline  solution  into  cuprous  oxide.  Treat  the 
dextrose  solution  with  about  -|-J  vol.  caustic  soda  and  then  carefully  add 
a  dilute  copper-sulphate  solution.  The  cupric  hydrate  is  thereby  dissolved, 
forming  a  beautiful  blue  solution,  and  the  addition  of  copper  sulphate 
is  continued  until  a  ver}^  small  amount  of  hydrate  remains  undissolved 
in  the  liquid.  This  is  now  warmed,  and  a  yellow  hydrated  suboxide  or  red 
suboxide  separates  even  below  the  boiling  temperature.  If  too  little  copper 
salt  has  been  added,  the  test  will  be  yello\\ish  brown  in  color,  as  in  Moore's 
test;  but  if  an  excess  of  copper  salt  has  been  added,  the  excess  of  hydrate 
is  converted  on  boiling  into  a  dark-brown  hydrate  which  interferes  with 
the  test.  To  prevent  the.se  difficulties  the  so-called  Fehlixg's  solution 
may  be  employed.  This  solution  is  obtained  by  mixing  just  before  use 
equal  volumes  of  an  alkaline  solution  of  Rochelle  salt  and  a  copper-sulphate 
solution  (see  Quantitative  Estimation  of  Sugar  in  the  Urine  in  regard  to 
concentration).  This  solution  is  not  reduced  or  noticeably  changed  by 
boiling.  The  tartrate  holds  the  excess  of  cupric  hydrate  in  solution,  iand  an 
excess  of  the  reagent  does  not  interfere  in  the  performance  of  the  test.  In 
the  presence  of  sugar  this  solution  is  reduced. 

BoTTGER-ALMiix's  tcst  is  based  on  the  property  dextrose  possesses  of 
redvicing  bismuth  oxide  in  alkaline  solution.  The  reagent  best  adapted  for 
this  purpose  is  obtained,  according  to  Nylander's  ^  modification  of  Alm^ix's 
original  test,  by  dissolving  4  grams  of  Rochelle  salt  m  100  parts  of  10  per 
cent  caustic-soda  solution  and  adding  2  grams  of  bismuth  subnitrate  and 
digesting  on  the  water-bath  until  as  much  of  the  bismuth  salt  is  dissolved 
as  possible.  If  a  dextrose  solution  is  treated  with  about  ^^  "^'ol-,  or  with  a 
larger  quantity  of  the  solution  when  large  quantities  of  sugar  are  present, 
and  boiled  for  a  few  minutes,  the  solution  becomes  first  j-ellow,  then  yel- 
lowish brown,  and  finally  nearly  black,  and  after  a  time  a  black  deposit 
of  bismuth  (?)  settles. 

The  property  of  dextrose  of  reducing  an  alkaline  solution  of  mercury 
on  boiling  is  the  basis  of  Knapp's  reaction  with  alkaline  mercuric  cyanide  and 
of  Sachsse's  reaction  with  an  alkaline  potassium-mercuric  iodide  solution. 

*  Zeitschr.  f.  physiol.  Chem.,  8. 


DEXTROSE.  117 

On  heating  with  phenylhydrazixe  acetate  a  dextrose  solution  gives  a 
precipitate  consisting  of  fine  yellow  crystalline  needles  which  are  nearly 
insoluble  in  water  but  soluble  in  boiling  alcohol,  and  which  separate  again 
on  treating  the  alcoholic  solution  with  water.  The  crj^stalline  precipitate 
consists  of  phenylglucosazone  (see  page  107).  This  compound  melts  when 
pure  at  204-205°  C.,.  dissolves  readily  in  pyridine  (0.25  gram  in  1  gram), 
and  precipitates  again  from  this  solution  as  crystals  on  the  addition  of 
benzene,  ligroin,  or  ether.  According  to  Neuberg  ^  this  behavior  can  be 
used  in  the  purification  of  the  osazone. 

Dextrose  is  not  precipitated  by  a  lead-acetate  solution,  but  is  almost 
completely  precipitated  by  a  solution  of  ammoniacal  basic  lead  acetate. 
On  warming,  the  precipitate  becomes  flesh-color  or  rose-red  (Rubner's 
reaction^). 

If  a  watery  solution  of  dextrose  is  treated  with  hcnzoylchloride  and 
an  excess  of  caustic  soda,  and  shaken  until  the  odor  of  benzoylchloride 
has  disappeared,  a  precipitate  of  benzoic-acid  ester  of  dextrose  will  be  pro- 
duced which  is  insoluble  in  water  or  alkali  (Baumaxx^). 

If  i-l  c.c.  of  a  dilute  water}^  solution  of  dextrose  is  treated  with  a  few 
drops  of  a  10  per  cent  alcoholic  solution  (free  from  acetone)  of  a-naphthol, 
the  liquid  is  colored  a  beautiful  violet  on  the  addition  of  1-2  c.c.  of  con- 
centrated sulphuric  acid  (Molisch).  According  to  Reixbold^  this 
reaction  depends  first  upon  the  formation  of  a  volatile  substance  which 
gives  a  bluish-violet  color  with  a-naphthol  and  sulphuric  acid  in  the  warmth. 
On  further  heating  furfurol  is  also  produced,  which  gives  a  raspberr}^- 
red  to  ruby-red  coloration. 

Diazoeenzexesulphonic  acid  gives  with  a  dextrose  solution  made  alkaline 
with  a  fixed  alkali  a  red  color,  which  after  10-15  minutes  gradually  changes  to 
violet.  Orthonitrophenylpropiolic  acid  yields  indigo  when  boiled  with  a  small 
quantity  of  dextrose  and  sodium  carbonate,  and  this  is  converted  into  indigo- 
white  by  an  excess  of  sugar.  An  alkaline  solution  of  dextrose  is  colored  deep 
red  on  being  warmed  with  a  dilute  solution  of  picric  acid.  The  behavior  of 
dextrose  towards  certain  pentose  reactions  has  already  been  given  on  page  112. 

A  more  complete  description  as  to  the  performance  of  these  several  tests 
will  be  given  in  detail  in  a  subsequent  chapter  (on  the  urine). 

Dextrose  is  prepared  pure  by  inverting  cane-sugar  by  the  following 
simple  method  of  Soxhlet  and  Tollexs,  being  a  modification  of  Schwarz's  ^ 
method : 

'Ber.  d.  d.  chem.  Gesellsch.,  32,  3384. 

2  Zeitschr.  f.  Biologic,  20. 

^  Ber.  d.  deutsch.  chem.  Gesellsch.,  19;  also  Kueny,  Zeitschr.  f.  physiol.  Chem.,  14, 
and  Skraup,  Wien.  Sitzungsber.,  98  (1888). 

'' Molisch,  Monatshefte  f.  Chem.,  7,  and  Centralbl.  f.  d.  med.  Wissensch.,  1887, 
pp.  34  and  49;    Reinbold,  Pfiiiger's  Arch.,  103. 

^Tollens,  Handbuch  der  Kohlehydrate,  2.  Aufl.  I,  39. 


118  THE  CARBOHYDRATES. 

Treat  12  litres  90  per  cent  alcohol  with  480  c.c.  fuming  hydrochloric 
acid  and  warm  to  45-50°  C. :  gradually  add  4  kilos  of  powdered  cane-sugar, 
and  allow  to  cool  after  two  hours,  when  all  the  sugar  will  have  dissolved 
and  teen  inverted.  To  incite  crystallization,  some  crystals  of  anhydrous 
dextrose  are  added,  and  after  several  days  the  crystals  are  sucked  dr}^  by 
the  air-pump,  washed  with  dilute  alcohol  to  remove  hydrochloric  acid,  and 
crystallized  from  alcohol  or  methyl  alcohol.  According  to  Tollens  it  is, 
best  to  dissolve  the  sugar  in  one  half  its  weight  of  water  on  the  water- 
bath  and  then  add  double  this  volume  of  90-95  per  cent  alcohol. 

In  detecting  dextrose  in  animal  fluids  or  extracts  of  tissues  we  may 
make  use  of  the  al)Ove-mentioned  reduction  tests,  the  optical  determination, 
fermentation,  and  phenylhydrazine  tests.  For  the  quantitative  estima- 
tion the  reader  is  referred  to  the  chapter  on  the  urine.  Those  liquids  contain- 
ing proteins  must  first  have  these  removed  by  coagulation  with  heat  and 
addition  of  acetic  acid,  or  by  precipitation  with  alcohol  or  metallic  salts, 
before  testing  for  dextrose.  In  regard  to  the  difficulties  of  operating  with 
l^lood  and  serous  fluids  we  refer  the  student  to  the  works  of  Schenck, 
RoHMAXN,  Abeli^s,  and  Seegen.^ 

The  guloses  are  stereoisomers  of  dextrose  and  may  be  prepared  artificially. 
.rf-Gulose  is  obtained  on  the  reduction  of  rf-gulonic  acid,  which  is  obtained  on  the 
reduction  of  glucuronic  acid. 

Mannoses.  —  d-Mannose,  also  called  seminose,  is  obtained  with  d-levulose 
on  the  careful  oxidation  of  rf-mannite.  It  is  also  obtained  on  the  hydrolysis 
of  natural  carbohydrates,  such  as  salep  sli  meand  reserve  cellulose  (especially 
from  the  shavings  from  the  ivory-nut).  It  is  dextrorotatory,  readily  fermente 
with  beer-yeast,  gives  a  hydrazone  not  readily  soluble  in  water,  and  an  osazone 
■"which  is  identical  with  that  from  d-glucose. 

Levulose,  also  called  ^-fructose  and  fruit-sugar,  occurs,  as  above 
stated,  mixed  with  dextrose  extensively  distributed  in  the  vegetable  king- 
»dom  and  also  in  honey.  It  is  formed  in  the  hydrolytic  cleavage  of  cane- 
.sugar  and  several  other  carbohydrates,  but  it  is  especially  readily  obtained 
'.by  the  hydrolytic  splitting  of  inulin.  In  extraordinary  cases  of  diabetes 
mellitus  we  find  levulose  in  the  urine.  Neuberg  and  Strauss  ^  have 
'detected  le\ailose  with  positiveness  in  human  blood-serum  and  exudates  in 
'Certain  cases. 

Le\ailose  crystallizes  with  difficulty  in  needles  partly  anhydrous  and 
partly  containing  water.  It  is  readily  soluble  in  water,  but  nearly  insoluble 
in  cold  absolute  alcohol,  though  rather  readily  in  boiling  alcohol.  Its 
: aqueous  solution  is  levogyrate.  Le\mlose  ferments  with  yeast,  and  gives 
the  same  reduction  tests  as  dextrose,  and  also  the  same  osazone.  It  gives 
a  compound  with  lime  which  is  less  soluble  than  the  corresponding  dex- 
trose compound.  Levulose  is  not  precipitated  by  sugar  of  lead  or  basic 
lead  acetate. 


'  Schenck,  Pfluger's  Arch.,  40  and  47;   Rohmann,  Centralbl.  f.  Physiol.,  4;  Abeles, 
2eitschr.  f.  physiol.  Chem.,  15;    Seegen,  Centralbl.  f.  Physiol,,  4. 

^  Zeitschr.  f.  physiol.  Chem.,  36,  which  also  contains  the  older  literature. 


LEVULOSE.  119 

L€\'iilose  does  not  reduce  copper  to  the  same  extent  as  dextrose. 
Under  similar  conditions  the  reduction  relationship  of  dextrose  to  le\'ulose 
is  100:92.08. 

In  detecting  le\'ulose  and  those  varieties  of  sugar  which  yield  le\'ulose 
on  cleavage  we  make  use  of  the  following  reaction  suggested  by  Seli- 
WANOFF.  To  a  few  cubic  centimetres  of  fummg  hydrochloric  acid,  add 
an  equal  volume  of  water  and  a  small  quantity  of  the  sugar  solution  or 
of  the  solid  substance  and  a  few  cr\-stals  of  resorcin  and  apply  heat.  The 
liquid  becomes  a  beautiful  red,  and  gradually  a  substance  precipitates 
which  is  red  in  color  and  soluble  in  alcohol.  According  to  Ofxer  ^  the 
nyxture  must  not  contain  more  than  12  per  cent  HCl,  and  the  boiling  must 
not  be  continued  longer  than  twenty  seconds,  otherwise  glucose,  mannose, 
and  indeed  maltose,  may  give  a  similar  reaction.  R.  and  0.  Adler^  per- 
form the  test  with  glacial  acetic  acid  and  a  drop  of  hydrochloric  acid  and 
some  resorcin,  in  which  case  a  reaction  with  aldoses  is  not  obtained.  Seli- 
waxoff's  reaction,  which  according  to  Rosix  may  be  made  more  delicate 
by  a  combination  with  the  spectroscopic  examination,  is,  as  Neuberg  ^  has 
shown,  a  general  reaction  for  ketoses. 

According  to  Neuberg,'*  methylphenylhydrazine  is  an  excellent  sub- 
stance to  vise  for  the  separation  and  detection  of  le\idose,  as  it  gives  a 
characteristic  levulose-methylphenylosazone.  This  osazone  when  recr^-s- 
tallized  from  alcohol  melts  at  153°.  It  shows  a  dextrorotation  of  1°  40' 
when  0.2  gram  of  the  osazone  is  dissolved  in  4  c.c.  pyridine  and  6  c.c. 
absolute  alcohol. 

Ofxer  has  made  objections  to  the  use  of  methylphenylhydrazine  in  the 
detection  of  levulose.  He  has  obtained  the  osazone  from  dextrose  and 
methjdphenylhydrazine,  although  the  osazone  is  formed  much  more  quickly 
with  le\n.ilose  than  with  dextrose.  Only  when  the  separation  of  the  osazone 
cr\'stals  with  methylphenylhydrazine  after  the  addition  of  acetic  acid 
takes  place  within  five  hours  at  ordinar}-  temperatures  is  the  presence  of 
le\T.ilose  positively  proven  (Ofxer  ^). 

The  use  of  secondary  asjTnmetric  hydrazines  as  a  general  reagent  for  ketoses 
and  as  a  means  of  separation  from  aldoses  is  objected  to  by  Ofxer. 

Levulose,  as  above  stated,  is  best  obtained  by  the  hydrolytic  cleavage 
of  inulin,  by  warming  with  faintly  acidulated  water. 

Sorbinose  (sorbin)  is  a  ketose  obtained  from  the  juice  of  the  berry  of  the 
mountain  ash  under  certain  conditions.  It  is  cr}'stalline  and  levogjTate,  and 
is  converted  into  sorbite  by  reduction. 

'  Monatshefte  f.  Chem.,  25. 

^  See  foot-note  5,  p.  112.  . 

'Zeitschr.  f.  physiol.  Chem.,  31;    Rosin,  ibid.,  3S. 

*  Ber.  d.  d.  chem.  Gesellsch.,  35;   also  Neuberg  and  Strauss,  ibid.,  36. 

^  Ibid.,  3",  and  Zeitschr.  f.  physiol.  Chem.,  45. 


120  THE  CARBOHYDRATES. 

Galactose  (not  to  be  mistaken  for  lactose  or  milk-sugar)  is  obtained 
on  the  hydrolytic  cleavage  of  milk-sugar  and  by  hydrolysis  of  many  other 
carbohydrates,  especially  varieties  of  gums  and  mucilaginous  bodies.  It 
is  also  obtained  on  heating  cerebrin,  a  nitrogenized  glucoside  prepared 
from  the  brain,  with  dilute  mineral  acids. 

It  crystallizes  in  needles  or  leaves  which  melt  at  168°  C.  It  is  some- 
what less  soluble  than  dextrose  in  water.  It  is  dextrogyrate  and  shows 
multirotation.  With  ordinary  yeast  the  galactose  is  slowly,  but  neverthe- 
less completely,  fermented.  It  is  fermented  by  a  great  variety  of  yeasts 
(E.  Fischer  and  Thierfelder),  but  not  by  Saccharomyces  apiculatus,- 
which  is  of  importance  in  physiological-chemical  investigations.  Galactose 
reduces  Fehling's  solution  to  a  less  extent  than  dextrose,  and  10  c.c. 
of  this  solution  are  reduced,  according  to  Soxhlet,  by  0.0511  gram  galac- 
tose in  1  per  cent  solution.  Its  phenylosazone  melts  at  193°  C,  and  is 
soluble  with  difficulty  in  water,  but  wdth  relative  ease  in  hot  alcohol. 
Its  solution  in  glacial  acetic  acid  is  optically  inactive.  In  the  test  with 
hydrochloric  acid  and  phloroglucin  galactose  gives  a  color  similar  to  the 
pentoses,  but  the  solution  does  not  give  the  absorption  spectrum.  On 
oxidation  it  first  yields  galactonic  acid  and  then  mucic  acid.  Both  I-  and 
?!-galactose  have  been  artificially  prepared. 

Talose  is  a  sugar  which  is  artificially  prepared  by  the  reduction  of  talonic 
acid.  Talonic  acid  is  obtained  from  d-galactonic  acid  by  heating  it  with  quinoline 
or  pyridme  to  140-150°  C. 

Appendix  to  the  Hexoses. 

CH2OH 
a -Glucosamine  2  (chitosamine),  C6Hi3N05=  Att  tsttt  ^,  whose  synthet- 

COH 

ical  preparation  has  already  been  given  on  page  108,  was  first  prepared  by 
Ledderhose  3  from  chitin  by  the  action  of  concentrated  hydrochloric  acid. 
Recently  it  has  been  obtained  as  a  cleavage  product  of  several  mucin 
substances  and  proteins  (see  pages  33  and  65).  Glucosamine  is,  as  E. 
Fischer  and  Leuchs"^  have  shown,  a  derivative  of  glucose  or  rf-mannose 
(probably  dextrose),  and  as  an  intermediary  member  between  the  hexoses 
and  the  oxyamino-acids  obtainable  from  the  proteins,  it  forms  in  certain 
regards  a  bridge  between  the  proteins  and  the  carbohydrates. 

The  free  base  is  readily  soluble  in  water  with  an  alkaline  reaction  and 

■See  F.  Voit,  Zeitschr.  f.  Biologie,  28  and  29. 

^  According  to  E.  Fischer's  suggestion  we  shall  use  the  term  glucosamine  instead 
of  the  term  chitosamine  which  has  lately  been  generally  used. 
^  Zeitschr.  f.  physiol.  Chem.,  2  and  4. 
^  Ber.  d.  d.  chem.  Gesellsch.,  36. 


GLUCOSAMINE.     GLUCURONIC  ACID.  121 

quickly  decomposes.  The  characteristic  hydrochloride  forms  colorless 
Ctystals  which  are  stable  in  the  air  and  readily  soluble  in  water,  difficultly 
soluble  in  alcohol,  and  insoluble  in  ether.  The  solution  is  dextrorotaton-, 
(a)D=+"0.15°  to  74.64°,  at  various  concentrations. ^  Glucosamine  has  a 
reducing  action  similar  to  glucose,  gives  the  same  osazone,  but  is  not  fer- 
mentable. With  benzoyl  chloride  and  caustic  soda  it  gives  a  cr\'stalline 
ester.  In  alkaline  solution  it  gives  with  phenylisocyanate  a  compound 
which  can  be  converted  into  its  anhydride  by  acetic  acid,  and  is  used  in 
the  separation  and  detection  of  glucosamine  (Steudel^).  On  oxidation 
with  nitric  acid  it  yields  norisosaccharic  acid,  whose  lead  salt  can  be 
separated  and  whose  salts  ^N^th  cinchonine  or  quinine  are  difficultly 
soluble  in  water  and  can  also  be  used  very-  successfully  in  the  detection 
of  glucosamine  (Neuberg  and  Wolff  3).  On  oxidation  with  bromine 
chitaminic  acid  (c?-glucosaminic  acid)  is  produced,  and  this  is  converted 
into  chitaric  acid,  CeHioOe,  b}'  nitrous  acid.  On  treatment  with  nitrous 
acid  glucosamine  yields  a  non-fermentable  sugar  called  chitose. 

Ehrlich  ■*  has  suggested  a  test  which  does  not  respond  with  the  free  glucos- 
amine, but  with  the  mucins  and  other  protein  bodies  containing  an  acetylated 
glucosamine.  It  consists  in  warming  the  substance,  which  has  previously  been 
treated  with  alkali,  with  a  hydrochloric-acid  solution  of  dimethylaminobenzalde- 
hyde,  when  a  beautiful  red  color  is  obtained. 

Glucosamine  is  best  prepared  from  decalcified  lobster-shells  by  treating 
with  hot  concentrated  hydrochloric  acid.^  In  regard  to  its  preparation 
from  protein  substances  we  must  refer  to  the  works  cited  on  page  33,  foot- 
note 1. 

Galactosamine  has  been  prepared  by  Schulz  and  Ditthorx  ^  from  a 
glucoproteid  of  the  spa\\"n  of  the  frog. 

CHO 

Glucuronic  acid  (glycuronic  acid),  C6Hio07=  (CH.0H)4,  is  a  derivative 

COOH 
of  dextrose  and  has  been  sjmt helically  prepared  by  E.  Fischer  and  Piloty  ' 
by  the  reduction  of  the  lactone  of  saccharic  acid.  On  oxidation  with 
bromine  it  forms  saccharic  acid,  and  on  reduction  it  yields  gidonic-acid 
lactone.  Salkowski  and  Neuberg  ^  have  obtained  /-xvdose  from  glu- 
curonic acid  by  splitting  off  CO2  by  means  of  putrefaction  bacteria. 

•  See  Hoppe-Seyler-Thierfelder's  Handbuch,  7.  Aufl.;  Sundwik,  Zeitschr.  f.  physiol. 
Chem.,  34. 

^  Zeitschr.  f.  physiol.  Chem.,  34. 

^  Ber.  d.  d.  chem.  Gesellsch..  34. 

^  Mediz.  Woche,  1901,  Xo.  15;  see  Langstein,  Ergebm'sse  der  Physiol.,  I,  Abt.  1,  SS. 

*  See  Hoppe-Seyler-Thierfelder's  Handbuch,  7.  Aufl. 
"Zeitschr.  f.  physiol.  Chem.,  29. 

'  Ber.  d.  d.  chem.  Gesellsch.,  24. 
^Zeitschi.  f.  phj'siol.  Chem.,  36. 


122  THE  CARBOHYDRATES. 

Glucuronic  acid  has  not  been  found  in  the  free  state  in  the  animal  body. 
It  occurs  to  a  slight  extent  in  normal  urine  as  a  conjugated  acid,  phenol- 
and  probably  also  indoxyl-  and  skatoxylglucuronic  acid  (Mayer  and 
Neuberg).  It  occurs  to  a  much  greater  extent  in  urine  as  conjugated 
acid  after  the  ingestion  of  certain  aromatic  and  also  aliphatic  substances, 
especially  camphor  and  chloral  hydrate.  It  was  obtained  first  by 
ScHMiEDEBERG  and  Meyer  from  camphoglucuronic  acid,  and  then  by 
V.  Merino  ^  from  urochloralic  acid  by  cleavage  with  dilute  acids.  Accord- 
ing to  P.  Mayer,2  on  the  oxidation  of  dextrose  a  partial  formation  of  glu- 
curonic acid  and  oxalic  acid  takes  place,  and  therefore,  according  to  him, 
an  increased  elimination  of  conjugated  glucuronic  acids  shows  in  certain 
cases  an  incomplete  oxidation  of  dextrose.  Conjugated  glucuronic  acids 
may  also  occur  in  the  blood  (P.  Mayer,  Lepine  and  Boulud^),  in 
the  faeces  and  in  the  bile.*  Neuberg  and  Neimann  ^  have  prepared 
certain  conjugated  glucuronic  acids  (see  Chapter  XV)  synthetically,  among 
them  being  euxanthic  acid.  The  most  abundant  source  of  glucuronic  acid 
is  the  artist's  pigment  "  Jaune  indien,"  which  contains  the  magnesium  salt 
of  euxanthic  acid  (euxanthon-glucuronic  acid). 

Glucuronic  acid  is  not  crystalline,  but  is  only  obtainable  as  a  syrup. 
It  dissolves  in  alcohol  and  is  readily  soluble  in  water.  If  the  aqueous  solu- 
tion is  boiled  for  an  hour  the  acid  is  partly  (20  per  cent)  converted  into 
the  crystalline  lactone,  glucurone,  CeHgOe,  which  is  soluble  in  water  and 
insoluble  in  alcohol.  The  alkali  salts  of  the  acid  are  cr>'stalline.  If  a 
concentrated  solution  of  the  acid  is  saturated  with  barium  hydrate  the 
basic  barium  salt  is  obtained  as  a  precipitate.  The  neutral  lead  salt  is 
soluble  in  water,  while  the  basic  salt  is  insoluble.  The  readily  crystallizable 
cinchonine  salt  can  be  used  in  isolating  glucuronic  acid  (Neuberg  ^). 
Glucuronic  acid  is  dextrorotatory,  while  the  conjugated  acids  are  levo- 
rotatory;  they  behave  like  dextrose  with  the  reduction  tests  and  do  not 
ferment  with  yeast.  They  give  the  pentose  reactions  with  phloroglucin 
or  orcin  and  hydrochloric  acid,  and  yield  abundant  furfurol  on  distillation 
with  hydrochloric  acid.  With  the  phenylhydrazine  test  they  give  crystal- 
line compounds  which  are  not  sufficiently  characteristic  (Thierfelder, 
P.  ]\Iayer'^).  By  the  action  of  3  mol.  phenylhydrazine  and  the  necessary' 
amount  of  acetic  acid  upon  1  mol.  glucuronic  acid  at  40°  for  a  few  days 


'  Mayer  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  29;  Schmiedeberg  u.  Meyer,  ibid., 
3;    V.  Mering,  ibid.,  6- 

2  Zeitschr.  f.  klin.  Med.,  47.     See  Chapter  XV. 

3  Zeitschr.  f.  physiol.  Chom.,  32;    Lepine  and  Boulud,  Compt.  rend.,  133,  134,  138. 
*  See  Bial,  Hofrnoister's  Beitrilge,  2,  and  v.  Leersum   ibid.,  3. 

'Zeitschr.  f.  physiol.  Chem.,  44. 

« Ber.  d.  d.  chem.  Gesellsch.,  33. 

'Thierfelder,  Zeitschr.  f.  physiol.  Chem.,  11,  13,  15;    P.  Mayer,  ibid.,  29. 


DISACCHARIDES.  123 

Neuberg  and  Neumann  obtained  the  glucuronic-acid  osazone,  which  was 
very  similar  to  glucosazone  and  melted  at  200-205°.  With  2>bromphenyl- 
hydrazine  hydrochloride  and  sodium  acetate,  glucuronic  acid  gives  7>brom- 
phenylhydrazine  glucuronate,  which  is  characterized  by  insolubility  in 
absolute  alcohol  and  by  a  very  prominent  levorotatory  action.  This  com- 
pound is  very  well  suited  for  the  detection  of  glucuronic  acid.^  Dissolved 
in  a  mixture  of  alcohol  and  pyridine  (0.2  gram  substance  in  4  c.c.  pyri- 
dine and  6  c.c.  alcohol)  the  rotation  is  7°  25',  which  corresponds  to 
(«)f=-369°. 

Glucuronic  acid  is  best  prepared  from  euxanthic  acid,  which  decomposes 
by  heating  it  with  water  to  120°  C.  for  several  hours.  The  filtrate  from 
the  euxanthon  is  concentrated  at  40°  C,  when  the  anhydride  gradually 
crystallizes  out.  On  boiling  the  mother-liquor  for  some  time  and  evaporat- 
ing further,  the  crystals  of  the  lactone  are  obtained.  In  regard  to  the 
quantitative  estimation  of  glucuronic  acid  we  must  refer  the  reader  to 
the  works  of  Tollens  and  his  collaborators  and  of  Neuberg  and  Neimann.^ 

Disaccharides. 

Some  of  the  varieties  of  sugar  belonging  to  this  group  occur  ready 
formed  in  nature.  Thus  we  have  saccharose  and  lactose.  Some,  on  the 
contrary,  such  as  maltose  and  isomaltose,  are  produced  by  the  partial 
hydrolytic  cleavage  of  complicated  carbohydrates.  Isomaltose  is  besides 
this  also  obtained  from  dextrose  by  reversion  (see  page  125). 

The  disaccharides  or  hexobioses  are  to  be  considered  as  anhydrides, 
derived  from  two  monosaccharides  with  the  exit  of  1  molecule  of  water. 
Corresponding  to  this,  their  general  formula  is  Ci2H220n.  On  hydrolytic 
cleavage  and  the  addition  of  water  they  yield  2  molecules  of  hexoses, 
either  2  molecules  of  the  same  hexose  or  one  each  of  two  different  hexoses. 
Thus 

Saccharose  +  H2O  =  dextrose  +  le\ailose ; 

Maltose     1  -1-  H2O  =  dextrose  -f-  dextrose ; 

Lactose       +  H2O  =  dextrose  -|-  galactose. 

The  levulose  turns  the  polarized  ray  more  to  the  left  than  the  dextro.se 
does  to  the  right;  hence  the  mixture  of  hexoses  obtained  on  the  cleavage  of 
saccharose  has  an  opposite  rotation  to  the  saccharose  itself.  On  this 
accovmt  the  mixture  is  called  invert-sugar,  and  the  hydrolytic  splitting 
is  designated  as  inversion.  This  term  inversion  is  not  only  used  for  the 
splitting  of  saccharose,  but  is  also   used  for  the  hydrolytic  cleavage  of 

'See  Neuberg,  Ber.  d.  d.  chem.  Gesellsch.,  32,  and  Mayer  and  Neuberg,  Zeitschr. 
f.  physiol.  Chem.,  29. 

^  Tollens,  Zeitschr.  f.  physiol.  Chem.,  44,  which  cites  also  the  older  work;  Neu- 
berg and  Neimann,  ibid.,  44;    Neuberg,  ibid.,  45. 


124  THE  CARBOHYDRATES. 

compound  sugars  into  monosaccharides.  The  reverse  reaction,  whereby 
monosaccharides  are  condensed  into  complex  carbohydrates,  is  called 
reversion. 

We  subdivide  the  disaccharides  into  two  groups:  first,  the  group  to 
which  saccharose  belongs,  where  the  members  do  not  have  the  property  of 
reducing  certain  metallic  oxides;  and  the  second  group,  to  which  tlie  two 
maltoses  and  lactose  belong,  the  members  acting  like  monosaccharides  in 
regard  to  the  ordinary  reduction  tests.  The  members  of  the  latter  group 
have  the  character  of  aldehyde  alcohols. 

Saccharose,  or  cane-sugar,  occurs  extensively  distributed  in  the  plant 
kingdom.  It  occurs  to  the  greatest  extent  in  the  stalk  of  the  sugar-millet 
and  sugar-cane,  the  roots  of  the  sugar-beet,  the  trunks  of  certain  varieties  of 
palms  and  maples,  in  carrots,  etc.  Cane-sugar  is  of  extraordinarily  great 
importance  as  a  food  and  condiment. 

Saccharose  forms  large,  colorless  monoclinic  crystals.  On  heating  it 
melts  in  the  neighborhood  of  160°  C,  and  on  heating  more  strongly  it  turns 
brown,  forming  so-called  caramel.  It  dissolves  very  readily  in  water,  and 
according  to  Scheibler  ^  100  parts  of  saturated  saccharose  solution  contain 
67  parts  of  sugar  at  20°  C.  It  dissolves  with  difficulty  in  strong  alcohol. 
Cane-sugar  is  strongly  dextrorotatory.  The  specific  rotation  is  only  slightly 
modified  by  concentration,  but  is  markedly  changed  by  the  presence  of 
other  inactive  substances.     The  specific  rotation  is  (a)D=  -1-66.5°. 

Saccharose  acts  indifferently  towards  Moore's  test  and  to  the  ordinary 
reduction  tests.  It  does  not  ferment  directly,  but  only  after  inversion, 
which  can  be  brought  about  by  an  enzyme  (invertin)  contained  in  the  yeast. 
An  inversion  of  cane-sugar  also  takes  place  in  the  intestinal  canal.  Con- 
centrated sulphuric  acid  blackens  cane-sugar  very  quickly  even  at  the 
ordinary  temperature,  and  anhydrous  oxalic  acid  does  the  same  on  warming 
on  the  water-bath.  Various  products  are  obtained  on  the  oxidation  of 
cane-sugar,  dependent  upon  the  variety  of  oxidizing  material  and  also  upon 
the  intensity  of  the  action.  Saccharic  acid  and  oxalic  acid  are  the  most 
important  products. 

The  reader  is  referred  to  complete  text-books  on  chemistry  for  the 
preparation  and  quantitative  estimation  of  cane-sugar. 

Maltose  (malt-sugar)  is  formed  in  the  hydrolytic  cleavage  of  starch  by 
malt  diastase,  saliva,  and  pancreatic  juice.  It  is  obtained  from  glycogen 
under  the  same  conditions  (see  Chapter  VIII).  Maltose  is  also  produced 
transitorily  in  the  action  of  sulphuric  acid  on  starch.  ]\laltose  forms  the 
fermentable  sugar  of  the  potato  or  grain  mash,  and  also  of  the  beerwort. 

^laltose  cr^^stallizes  with  1  molecule  water  of  crystallization  in  fine 
white  needles.     It  is  readily  soluble  in  water,  rather  easily  in  alcohol,  but 

'See  Tollens'  Handbuch  der  Kohlehydrate,  2,  Atifl.  1,  124. 


ISOMALTOSE.    LACTOSE.  125 

insoluble  in  ether.  Its  solutions  are  dextrorotatory;  and  the  specific- 
rotation  is  variable,  depending  upon  the  concentration  and  temperature, 
but  is  considerably  stronger  than  dextrose.^  Maltose  ferments  readily  and 
completely  with  yeast,  and  acts  like  dextrose  in  regard  to  the  reduction 
tests.  It  yields  phenylmaltosazone  on  warming  with  phenylhydrazine  for 
1-^-  hours.  This  phenylmaltosazone  melts  at  206°  C.  and  is  more  soluble 
than  the  glucosazone.  Maltose  differs  from  dextrose  chiefly  in  the  follow- 
ing: It  does  not  dissolve  as  readily  in  alcohol,  has  a  stronger  dextrorota- 
tory power,  and  has  a  feebler  reducing  action  on  Fehling's  solution.  10 
c.c.  Fehling's  solution  is,  according  to  Soxhlet,^  reduced  by  77.8  milli- 
grams anhydrous  maltose  in  approximately  1  per  cent  solution. 

Isomaltose.  This  variety  of  sugar,  as  has  been  shown  by  Fischer,^  is 
produced,  besides  dextrin-like  products,  by  reversion  and  by  the  action  of 
fuming  hydrochloric  acid  on  dextrose.  A  re-formation  of  isomaltose  and 
other  sugars  from  dextrose  can  also  be  brought  about  by  means  of  yeast 
maltase  (Hill  and  Emmerlinc*).  It  is  also  formed,  besides  ordinary 
maltose,  in  the  action  of  diastase  on  starch  paste,  and  occurs  in  beer  and 
in  commercial  starch-sugar.  The  formation  of  isomaltose  in  the  hydrolysis 
of  starch  by  malt  diastase  has  been  denied  by  many  investigators  because 
they  considered  isomaltose  as  contaminated  maltose.^  It  is  also  produced, 
with  maltose,  by  the  action  of  saliva  or  pancreatic  juice  (KtJLZ  and  Vogel) 
or  blood-serum  (Rohmann  ^)  on  starch. 

Isomaltose  dissolves  very  readily  in  water,  has  a  pronounced  sweetish 
taste,  and  does  not  ferment,  or,  according  to  some,  only  very  slowly.  It 
is  dextrorotatory,  and  has  very  nearly  the  same  power  of  rotation  as  mal- 
tose. Isomaltose  is  characterized  by  its  osazone.  This  forms  fine  yellow 
needles,  which  begin  to  form  drops  at  140°  C.  and  melt  at  150-153°  C. 
It  is  rather  easily  soluble  in  hot  water  and  dissolves  in  hot  absolute  alcohol 
much  more  readily  than  the  maltosazone.  Isomaltose  reduces  copper  as 
well  as  bismuth  solutions. 

Lactose  (milk-sugar).  As  this  sugar  occurs  exclusively  in  the  animal 
world,  in  the  milk  of  human  beings  and  animals,  it  will  be  treated  in  a 
following  chapter  (on  milk). 

*  See  Hoppe-Seyler-Thierfelder's  Handbuch,  7.  Aufl. 

^  Cited  from  Tollens'  Handbuch  der  Kohlehydrate,  2.  Aufl.  1,  154. 

^  Ber,  d.  deutsch.  chem.  Gesellsch.,  23  and  28. 

■"Emmerling,  ibid.,  34;    Hill,  ibid.,  34,  and  1.  c.,  foot-note  1,  p.  16. 

^  Brown  and  Morris,  Journ.  of  Chem.  Soc.,  1895;  Chem.  News,  72.  See  also  Ost. 
Ulrich,  and  Jalowetz,  Ref.  in  Ber.  d.  deutsch.  chem.  Gesellsch.,  28;  Ling  and  Baker, 
Journ.  of  Chem.  Soc,  1895;    Pottevin,  Chem.  Centralbl.,  1899,  II,  1023. 

°  Kiilz  and  Vogel,  Zeitschr.  f.  Biologic,  31;  Rohmann,  Centralbl.  f.  d.  med.  Wis- 
sensch.,  1893,  849. 


126  THE  CARBOHYDRATES. 

Polysaccharides. 

If  we  exclude  the  hexotrioses  and  the  few  remaining  sugar-Uke  poly- 
saccharides, this  group  includes  a  great  number  of  very  complex  carbo- 
hydrates, which  occur  only  in  the  amorphous  condition,  or  at  least  not  as 
crystals  in  the  ordmary  sense.  Unlike  the  bodies  belonging  to  the  other 
groups,  these  have  no  sweet  taste.  Some  are  soluble  in  water,  while  others 
swell  up  therein,  especially  in  warm  water,  and  finally  are  neither  dissolved 
nor  visibly  changed.  Polysaccharides  are  ultimately  converted  into  mono- 
saccharides by  hydrolytic  cleavage. 

The  polysaccharides  (not  sugar-like)  are  ordinarily  divided  into  the 
following  chief  groups:  starch  group,  gum  and  vegetable-mucilage  group, 
and  cellulose  group. 

Starch  Group,  (C6H]o05)x. 

Starch,  amylum,  (C6Hio05)x.  This  substance  occurs  in  the  plant  king- 
dom very  extensively  distributed  in  the  different  parts  of  the  plant,  espe- 
cially as  reserve  food  in  the  seeds,  roots,  tubers,  and  trunks. 

Starch  is  a  white,  odorless,  and  tasteless  powder,  consisting  of  small 
granules  which  have  a  stratified  structure  and  different  shape  and  size  in 
different  plants.  According  to  the  ordinar}^  opinion  the  starch  granules 
consist  of  two  different  substances,  starch  granulose  and  starch  cellu- 
lose (v.  Nageli),  corresponding  to  ]\Iaquexne  and  Roux's  ^  amylose  and 
amylopectin,  of  which  the  first  alone  is  converted  into  sugar  on  treatment 
with  diastatic  enzymes. 

Starch  is  considered  insoluble  in  cold  water.  The  grains  swell  up  in 
warm  water  and  burst,  yielding  a  paste.  Starch  is  insoluble  in  alcohol  and 
ether.  On  heating  starch  with  water  alone,  or  heating  with  glycerine  to 
190°  C,  or  on  treating  the  starch  grains  with  6  parts  dilute  hydrochloric 
acid  of  sp.  gr.  1.07  at  ordinary  temperature  for  six  to  eight  weeks,^  it  is 
converted  into  soluble  starch  (amylodextrin,  amidulin).  Soluble  starch 
is  also  formed  as  an  intermediate  step  in  the  conversion  of  starch  into  sugar 
by  dilute  acids  or  diastatic  enzymes.  Soluble  starch  may  be  precipitated 
from  very  dilute  solutions  by  baryta-water.^ 

Starch  granules  swell  up  and  form  a  pasty  mass  in  caustic  potash  or 
soda.  This  mass  gives  neither  ^Ioore's  nor  Trommer's  test.  Starch 
paste  does  not  ferment  with  yeast.     The  most  characteristic  test  for  starch 

*  V.  Nageli,  Botan.  Mitteil,,  1863;  Maquenne  and  Roux,  Compt.  rend.,  140,  and 
Bull.  Soc,  chim.  de  Paris  (3),  33. 

^  See  ToUens'  Handb.,  191.  In  regard  to  other  methods,  see  Wroblewsky,  Ber.  d. 
deutsch.  chem.  Gesellsch.,  30;    Syniewski,  ibid. 

^  In  regard  to  the  compounds  of  soluble  starch  and  dextrins  with  barium  hydrate, 
see  Biilow,  Ffliiger's  Arch.,  62. 


STARCH.     INULIN.  127 

is  the  blue  coloration  produced  by  iodine  in  the  presence  of  hydriodic 
acid  or  alkali  iodides.^  This  blue  coloration  disappears  on  the  addition  of 
alcohol  or  alkalies,  and  also  on  warming,  but  reappears  again  on  cooling. 

On  boiling  with  dilute  acids  starch  is  converted  into  dextrose.  In  the 
conversion  by  means  of  diastatic  enzymes  we  have  as  a  rule,  besides  dextrin, 
maltose,  and  isomaltose,  only  very  little  dextrose.  We  are  considerably  in 
the  dark  as  to  the  kind  and  number  of  intermediate  products  produced  in 
this  process  (see  Dextrins). 

Starch  may  be  detected  by  means  of  the  microscope  and  by  the  iodine 
reaction.  Starch  is  quantitatively  estimated,  according  to  Sachsse's 
met  hod, 2  by  converting  it  into  dextrose  by  hydrochloric  acid  and  then 
determining  the  dextrose  by  the  ordinary  methods. 

Inulin,  (C6Hio05)x  +  H20,  occurs  in  the  underground  parts  of  many 
compositiE,  especially  in  the  roots  of  the  Inula  helenium,  the  tubers  of  the 
dahlia,  the  varieties  of  helianthus,  etc.  It  is  ordinarily  obtained  from  the 
tubers  of  the  dahlia. 

Inulin  forms  a  white  powder  similar  to  starch,  consisting  of  spheroid 
crystals  which  are  readily  soluble  in  warm  water,  without  forming  a  paste. 
It  separates  slowly  on  cooling,  but  more  rapidly  on  freezing.  Its  solutions 
are  levogyrate  and  are  precipitated  by  alcohol,  and  are  colored  only  yellow 
with  iodine.  Inulin  is  converted  into  the  levogyrate  monosaccharide  levu- 
lose  on  boiling  with  dilute  sulphuric  acid.  Diastatic  enzymes  have  no  or 
only  very  slight  action  on  inulin .^ 

According  to  Dean  ^  inulin  occurs  together  with  other  substances,  levulins, 
which  are  more  soluble  and  have  less  rotation.  He  suggests  that  we  limit  the 
name  inulin  to  that  of  carbohydrate  (or  mixture  of  carbohydrates),  which  is 
readily  precipitable  by  60  per  cent  alcohol  and  shows  a  specific  rotation  of 
(«)d= -38-40°. 

Lichenin  (moss-starch)  occurs  in  many  lichens,  especially  in  Iceland  moss. 
It  is  not  soluble  in  cold  water,  but  swells  up  into  a  jelly.  It  is  soluble  in  hot 
water,  forming  a  jelly  on  allowing  the  concentrated  solution  to  cool.  It  is  colored 
yellow  by  iodine  and  yields  glucose  on  boiling  with  dilute  acids.  Lichenin  is 
not  changed  by  diastatic  enzymes  such  as  ptyalin  or  amylopsin  (Nilson  ^). 

Glycogen.  This  carbohydrate,  which  stands  to  a  certain  extent  between 
starch  and  dextrin,  is  principally  found  in  the  animal  kingdom,  hence  it 
will  be  considered  in  a  subsequent  chapter  (on  the  liver). 

'  See  Mylius,  Ber.  d.  deutsch.  chem.  Gesellsch.,  20,  and  Zeitschr.  f.  physiol.  Chem.,  11. 

2Tollens'  Handb.,  2.  Aufl.  1,  187. 

Ubid.,  208. 

^  Amer.  Chem.  Journ.,  32. 

'Upsala  Lakaref.  Forh.,  28. 


128  THE  CARBOHYDRATES. 

The  Gums  and  Vegetable  Mucilages,   (C6Hio05)x. 

These  bodies  may  be  di^'ided  into  two  chief  groups,  accordmg  to  their 
origin  and  occurrence,  namely,  the  dextrin  group  and  the  vegetable  gums  or 
mucilages.  The  dextrins  stand  in  close  relationsliip  to  the  starches  and 
are  formed  therefrom  as  intermediate  products  in  the  action  of  acids  and 
diastatic  enzymes.  The  various  kinds  of  vegetable  gums  and  vegetable 
mucilages  occur,  on  the  contrar}^,  as  natural  products  in  the  vegetable 
kingdom,  and  some  may  be  separated  from  certain  plants  as  amorphous, 
transparent  masses,  and  others  may  be  extracted  from  certain  parts  of  the 
plant,  such  as  the  wood  and  seeds,  by  proper  solvents. 

The  dextrins  yield  as  final  products  only  hexoses,  indeed  only  dex- 
trose, on  complete  hydrolysis.  The  vegetable  gums  and  the  mucilages 
yield,  on  the  contrarj%  not  only  hexoses,  but  also  an  abundance  of  pentoses 
(gum  arabic  and  wood-gum).  d-Galactose  occurs  often  among  the  hexoses, 
and  accordingly  as  a  differentiation  from  the  dextrins,  they  yield  mucic 
acid  on  oxidation  with  nitric  acid.  The  dextrins,  as  svell  as  the  ordinary 
varieties  of  gums  and  mucilages,  are  precipitated  by  alcohol.  Basic  lead 
acetate  precipitates  the  gums  and  mucilages,  but  not  the  dextrins. 

Dextrin  (starch-gum,  British  gum)  is  produced  on  heating  starch  to 
200-210°  C,  or  by  heating  starch,  which  has  previously  been  moistened 
with  water  containing  a  little  nitric  acid,  to  100-110°  C.  Dextrins  are  also 
produced  by  the  action  of  dilute  acids  and  diastatic  enzymes  on  starch. 
We  are  not  quite  clear  in  regard  to  the  steps  taking  place  in  the  above 
processes,  but  the  ordinary  views  are  as  follows:  The  first  product,  which 
gives  a  blue  with  iodine,  is  soluble  starch  or  amylodextrin,  which  on  further 
hydrolytic  cleavage  yields  sugar  and  erythrodextnn,  which  is  colored  red 
by  iodine.  On  further  cleavage  of  this  erj-throdextrin  more  sugar  and 
a  dextrin,  achroodextrin,  which  is  not  colored  by  iodine,  are  formed.  From 
this  achroodextrin  after  successive  splittings  we  have  sugar  and  dextrins 
of  lower  molecular  weights  formed,  until  finally  we  have  sugar  and  a  dextrin, 
maltodextrin,  which  refuses  to  split  further,  as  final  products.  The  views 
are  rather  contradictory  in  regard  to  the  number  of  dextrins  which  occur 
as  intermediate  steps.  The  sugar  formed  is  isomaltose,  from  which  mal- 
tose and  only  verv'  little  dextrose  are  produced.  Another  \'iew  is  that 
first  several  dextrins  are  formed  consecutively  in  the  successive  splittings, 
with  hydration,  and  then  finally  the  sugar  is  formed  by  the  splitting  of 
the  last  dextrin.  According  to  Moreau,  in  the  first  stages  of  saccharifica- 
tion  amylodextrin,  ery^throdextrin,  achroodextrin  and  sugar  are  formed 
simultaneously.  Other  investigators,  especially  Syniewski,  have  recently 
suggested  other  views  on  this  subject.^ 


'  In  regard  to  the  various  views  on  the  theories  of  the  saccharification  of  starch, 


CELLULOSE.  129 

The  various  dextrins  have  not  as  yet  been  separated  from  each  other, 
nor  isohited  as  chemical  individuals.  Recently  Youxo  ^  has  tried  their 
separation  by  means  of  neutral  salts,  especially  ammonium  sulphate,  and 
MoREAU  by  the  aid  of  a  bar\-ta-alcohol  method.  We  cannot  enter  into 
the  differences  as  to  the  dextrins  so  separated,  and  only  the  characteristic 
properties  and  reactions  will  be  given  for  the  dextrins  in  general. 

The  dextrms  appear  as  amorphous,  white  or  yellowish-white  powders 
which  are  readily  soluble  in  water.  Their  concentrated  solutions  are  \'iscid 
and  stick}',  similar  to  gum  solutions.  The  dextrins  are  dextrog}'rate. 
They  are  insoluble  or  nearly  so  in  alcohol,  and  insoluble  in  ether.  Waters- 
solutions  of  dextrins  are  not  precipitated  by  basic-lead  acetate.  Dextrins 
dissolve  cupric  hydrate  in  alkaline  liquids,  forming  a  beautiful  blue  solu- 
tion, which,  as  is  generally  admitted,  is  reduced  by  pure  dextrins.  Accord- 
ing to  !\IoREAU  pure  dextrin  has  no  reducing  action.  The  dextrins  are  not 
directly  fermentable. 

The  vegetable  gums  are  soluble  in  water,  forming  solutions  which  are  viscid 
but  may  be  filtered.  We  designate,  on  the  contrary,  as  vegetable  mucilages 
those  varieties  of  gum  which  do  not  or  onh'  partly  dissolve  in  water,  and  which 
swell  up  therein  to  a  greater  or  less  extent.  The  natural  varieties  of  gum  and 
mucilage,  to  which  belong  several  generally  known  and  important  substances, 
such  as  gum  arable,  wood-gum,  cherry-gum,  salep,  and  Cjuince  mucilage,  and 
probably  also  the  little-studied  pectin  substances,  will  not  be  treated  in  detail, 
because  of  their  unimportance  from  a  physiological  standpoint. 

The  Cellulose  Group,  'C6Hio05)x. 

Cellulose  is  that  carbohydrate,  or  perhaps  more  correctly  mixture  of 
carbohydrates,  which  forms  the  chief  constituent  of  the  walls  of  the  plant- 
cells.  This  is  true  for  at  lea.st  the  walls  of  the  young  cells,  while  in  the 
walls  of  the  older  cells  the  cellulose  is  extensively  incmsted  with  a  sub- 
stance called  LiGxix. 

The  true  celluloses  are  characterized  by  their  great  insolubility.  They 
are  insoluble  in  cold  or  hot  water,  alcohol,  ether,  dilute  acids,  and  alkalies. 
We  have  only  one  specific  solvent  for  cellulose,  and  that  is  an  ammoniacal 
solution  of  copper  oxide  called  Schweitzer's  reagent.  The  cellulose  may 
be  precipitated  from  this  solvent  by  the  addition  of  acids,  and  obtained  as 
an  amorphous  powder  after  washing  with  water. 

Cellulose  is  converted  into  a  substance,  so-called  amyloid,  which  gives 
a  blue  coloration  with  iodine  by  the  action  of  concentrated  sulphuric  acid. 

see  Musculus  and  Gruber,  Zeitschr.  f.  physiol.  Chem.,  2;  Lintner  and  Didl,  Ber.  d.  d. 
chem.  Gesellsch.,  26  and  2S;  Biilow,  1.  c;  Brown  and  Heron,  Journ.  of  Chem.  Soc,  1879; 
Brown  and  Morris,  ibid.,  1885  and  1889;  Moreau,  Biochem.  Centralbl.,  3,  648;  Sy- 
niewski,  Annal.  d.  Chem.  u.  Pharm.,  309,  and  Chem.  Centralbl.,  1902,  2. 

*  Journ.  of  Physiol.,  22,  which  contains  the  older  researches  of  Nasse,  Kriiger, 
Neumeister,  Pohl,  and  Halliburton.     Moreau,  1.  c. 


130  THE  CARBOHYDRATES. 

B}^  the  action  of  strong  nitric  acid  or  a  mixture  of  nitric  acid  and  concen- 
trated sulphuric  acid  celluloses  are  converted  into  nitric-acid  esters  or  nitro- 
celluloses,  which  are  highly  explosive  and  have  found  great  practical  use. 

The  ordinary  celluloses  when  treated  at  the  ordinary  temperature  with 
strong  sulphuric  acid  and  then  boiled  for  some  time  after  diluting  with 
water  are  converted  into  dextrose.  We  also  have  celluloses  which  behave 
differently,  namely,  those  which  yield  mannose  on  the  above  treatment. 

Hemicelluloses  are,  according  to  E.  Schui-ze,  those  constituents  of  the 
cell-wall  related  to  cellulose  which  differ  from  the  ordinary  cellulose  by  dissolv- 
ing on  heating  with  strongly  diluted  mineral  acids,  such  as  1.25  per  cent  sulphuric 
acid,  and  of  yielding  arabinose,  xylose,  galactose,  and  mannose  instead  of  dextrose. 
The  hemicelluloses  (from  lupin  seeds)  are  hydrolized  even  by  0.1  per  cent  hydro- 
chloric acid  and  are  dissolved,  although  only  slowly,  by  diastatic  enzymes 
(ScHULZE  and  Castoro  ')• 

The  cellulose,  at  least  in  part,  undergoes  decomposition  in  the  infest iral 
tract  of  man  and  animals.  A  closer  discussion  of  the  nutritive  value  of 
cellulose  will  be  given  in  a  future  chaj^ter  (on  digestion).  The  great  im- 
portance of  the  carbohydrates  in  the  animal  economy  and  to  animal  metab- 
olism will  also  be  given  in  the  following  chapters. 


'  E.  Schulze,  Zeitschr.  f.  physiol.  Chem.,  16  and  19,  with  Castoro,  ibid.,  36. 


CHAPTER  IV. 
THE  ANIMAL  FATS. 

The  fats  form  the  third  chief  group  of  the  organic  food  of  man  and 
animals.  They  occur  very  widely  distributed  in  the  animal  and  plant 
kingdoms.  Fat  occurs  in  all  organs  and  tissues  of  the  animal  organism, 
though  the  quantity  may  be  so  variable  that  a  tabular  exhibit  of  the  amount 
of  fat  in  different  organs  is  of  little  interest.  The  marrow  contains  the 
largest  quantity,  having  over  96  per  cent.  The  three  most  important 
deposits  of  fat  in  the  animal  organism  are  the  intermuscular  connective 
tissue,  the  fatty  tissue  in  the  abdominal  cavity,  and  the  subcutaneous  con- 
nective tissues.  In  plants,  the  seeds  and  fruit  and  in  certain  instances 
also  the  roots,  are  rich  in  fat. 

The  fats  consist  almost  entirely  of  so-called  neutral  fats  with  only  very 
small  quantities  of  fatty  acids.  The  neutral  fats  are  esters  of  the  triatomic 
alcohol,  glycerine,  with  monobasic  fatty  acids.  These  esters  are  triglycerides, 
that  is,  the  hydrogen  atoms  of  the  three  hydroxyl  groups  of  the  glycerine 
are  replaced  by  the  fatty-acid  radicals,  and  their  general  formula  is  there- 
fore C3H5O3.R3.  The  animal  fats  consist  chiefly  of  esters  of  the  three  fatty 
acids,  stearic,  palmitic,  and  oleic  acids.  In  certain  fats,  especially  in  milk- 
fat,  glycerides  of  fatty  acids  such  as  butyric,  caproic,  caprylic,  and  capric 
acids  also  occur  in  considerable  amounts.  Besides  the  above-mentioned 
ordinaiy  fatty  acids,  stearic,  palmitic,  and  oleic  acids,  we  also  find  in  human 
and  animal  fat,  exclusive  of  certain  fatty  acids  only  little  studied,  the  fol- 
lowing non-volatile  fatty  acids,  as  glycerides,  namely,  lauric  acid,  C12H24O2, 
myristic  acid,  C14H28O2,  and  arachidic  acid,  C20H40O2.  In  the  plant  king- 
dom triglycerides  of  other  fatty  acids,  such  as  lauric  acid,  myristic  acid, 
linoleic  acid,  erucic  acid,  etc.,  sometimes  occur  abundantly.  Besides  these, 
oxyacids  and  high  molecular  alcohols  have  been  found  in  many  plant  fats. 
The  extent  to  which  traces  of  these  oxyacids  occur  in  the  animal  kingdom  has 
not  been  thoroughly  investigated,  but  the  occurrence  of  monoxystearic  acid 
seems  to  have  been  proven.^     The  occurrence  of  high  molecular  alcohols, 

'  Erben,  Zeitschr.  f.  physiol.  Chem.,  30;   Bernert,  Arch.  f.  exp.  Path.  u.  Pharm.,  49. 

131 


132  THE  ANIMAL  FATS. 

althoii,2;h  ordinarily  only  in  small  amounts,  has  on  the  contrary  been  posi- 
tively shown  in  animal  fat. 

The  animal  fats  are  of  the  greatest  interest  and  consist  of  a  mixture  of 
varying  quantities  of  tristearin,  tripalmitin,  and  triolein,  having  an 
average  elementary  composition  of  C  76.5,  H  12.0,  and  O  11.5  per  cent. 
It  must  be  remarked  that  in  animal  fat  (mutton  and  beef  tallow)  as  well 
as  in  plant  fat  (olive-oil)  mixed  triglycerides,  such  as  dipalmityl-olein, 
distearyl-palmitin  and  distearyl-olein,  occur  and  that  these  mixed  glycerides 
may  also  be  prepared  synthetically.^ 

Fats  from  different  species  of  animals,  and  even  from  different  parts  of 
the  same  animal,  have  an  essentially  different  consistency,  depending  upon 
the  relative  amounts  of  the  different  individual  fats  present.  In. solid  fats 
— as  tallow — tristearin  and  tripalmitin  are  in  excess,  while  the  less  solid 
fats  are  characterized  by  a  greater  abundance  of  tripalmitin  and  triolein. 
This  last-mentioned  fat  is  found  in  greater  quantities  proportionally  in 
cold-blooded  animals,  and  this  accounts  for  the  fact  that  the  fat  of  these 
animals  remains  fluid  at  temperatures  at  which  the  fat  of  warm-blooded 
animals  solidifies.  Human  fat  from  different  organs  and  tissues  contains, 
in  full  numbers,  67-85  per  cent  triolein .2  The  melting-point  of  different 
fats  depends  upon  the  composition  of  the  mixtures,  and  it  not  only  varies 
for  fat  from  different  tissues  of  the  same  animal,  but  also  for  the  fat  from 
the  same  tissues  in  various  kinds  of  animals. 

Neutral  fats  are  colorless  or  yellowish  and,  when  perfectly  pure,  odorless 
and  tasteless.  They  are  lighter  than  water,  on  which  they  float  when  in  a 
molten  condition.  They  are  insoluble  in  water,  dissolve  in  boiling  alcohol, 
but  separate  on  cooling — often  in  crystals.  They  are  easily  soluble  in 
ether,  benzene,  and  chloroform.  The  fluid  neutral  fats  give  an  emulsion 
when  shaken  \\ith  a  solution  of  gxim  or  albumin.  With  water  alone  they 
give  an  emulsion  only  after  vigorous  and  prolonged  shaking,  but  the 
emulsion  is  not  persistent.  The  presence  of  some  soap  causes  a  very  fine 
and  permanent  emulsion  to  form  easily.  Fat  produces  spots  on  paper 
which  do  not  disappear;  it  is  not  volatile;  it  boils  at  about  300°  C.  with 
partial  decomposition,  and  bums  with  a  luminous  and  smoky  flame.  The 
fatty  acids  have  most  of  the  above-mentioned  properties  in  common  with 
the  neutral  fats,  but  differ  from  them  in  being  soluble  in  alcohol-ether,  in 
having  an  acid  reaction,  and  by  not  giving  the  acrolein  test.  The  neutral 
fats  generate  a  strong  irritating  vapor  of  acrolein,  due  to  the  decomposition 
of  glycerine,   C3H5(0H)3  — 2H20  =  C3H40,   when   heated   alone,   or  more 

*Guth,  Zeitschr.  f.  Biologie,  44;  W.  Hansen,  Arch.  f.  Hygiene,  42;  Holde  and 
Stange,  Ber.  d.  d.  chem.  Gesellsch.,  34;   Kreis  and  Hafner,  ibid.,  36. 

^  See  Knopfelmacher,  "Untersuch.  iiber  das  Fett  im  Sauglingsalter,"  etc.,  Jahrbuch 
f.  Kinderheilkunde  (N.  F.),  45,  which  also  contains  the  older  literature;  Jaeckle, 
Zeitschr.  f.  physiol.  Chem.,  36. 


STEARIN.  133 

easily  when  heated  with  potassium  bisulphate  or  with  other  dehydrating 
substances. 

The  neutral  fats  may  be  spUt  by  the  addition  of  the  constituents  of 
water  according  to  the  following  equation :  C3H5(OR)3  +  3HoO  =  C3H5(OH)3 
+  3H0R.  This  splitting  may  be  produced  by  the  pancreatic  enzyme  and 
other  enzymes  occurring  in  the  animal  and  vegetable  kingdoms,  or  by 
superheated  steam.  We  most  frequently  decompose  the  neutral  fats  by 
boiling  them  with  not  too  concentrated  caustic  alkali,  or,  still  better  (in 
biochemical  researches),  with  an  alcoholic  potash  solution  or  -uith  sodium 
alcoholate.  By  this  procedure,  which  is  called  saponification,  the  alkali 
salts  of  the  fatty  acids  (soaps)  are  formed.  If  the  saponification  is  made 
with  lead  oxide,  then  lead  plaster,  the  lead  salt  of  the  fatty  acids,  is  pro- 
duced. By  saponification  is  to  be  understood  not  only  the  cleavage  of 
neutral  fats  by  alkalies,  but  also  the  splitting  of  neutral  fats  into  fatty 
acids  and  glycerine  in  general. 

On  keeping  fats  for  a  long  time  in  contact  with  air  they  undergo  a  change, 
becoming  yellow  in  color  and  acid  in  reaction,  and  they  develop  an 
unpleasant  odor  and  taste,  becoming  rancid.  In  this  change  a  part  of  the 
fat  is  split  mto  fatty  acids  and  gl3'cerine,  and  then  an  oxidation  of  the 
free  fatty  acids  takes  place,  producing  volatile  bodies  of  an  unpleasant 
odor. 

The  three  most  important  fats  of  the  animal  kmgdom  are  stearin, 
palmitin.  and  olein. 

CH2O.C18H35O 

Stearin  or  tristearm,  CoyHnoOe^CHO.CisHsoO,  occurs  especially  in 

CH2O.C18H30O 
the  solid  varieties  of  tallows,  but  also  in  the  vegetable  fats.  Stearic  acid, 
C18H36O2,  is  foimd  in  the  free  state  in  decomposed  pus,  in  the  expectora- 
tions in  gangrene  of  the  lungs,  and  in  cheesy  tuberculous  masses.  It 
occurs  as  lime  soap  in  excrements  and  adipocere,  and  in  this  last  product 
also  as  an  ammonium  soap.  It  also  exists  as  alkali  soap  in  the  blood,  bile, 
transudations  and  pus,  and  in  the  urine  to  a  slight  extent. 

Stearin  is  the  hardest  and  most  insoluble  of  the  three  ordinary'  neutral 
fats.  It  is  nearly  insoluble  in  cold  alcohol,  and  soluble  "U'ith  great  difficult}' 
in  cold  ether  (225  parts).  It  separates  from  warm  alcohol  on  coolmg  as 
rectangular,  less  frequenth^  as  rhombic  plates.  The  statements  in  regard 
to  the  melting-point  are  somewhat  varied.  Rire  stearin,  accordmg  to 
Heixtz.i  melts  transitority  at  55°  and  permanently  at  71.5°.  The  stearin 
from  the  fatty  tissues  (not  pure)  melts  at  63°  C. 
CH3 

Stearic  acid,  (CH2)i6.  cr\'stallizes  (on  cooling  from  boiling  alcohol)  m 
COOH 

*  Annal.  d.  Chem.  u.  Pharm.,  92 


134  THE  ANIMAL  FATS. 

large,  shining,  long  rhombic  scales  or  plates.  It  is  less  soluble  than  the 
other  fatty  acids  and  melts  at  69.2°  C.  Its  barium  salt  contains  19.49  per 
cent  barium,  and  its  silver  salt  contains  27.59  per  cent  silver. 

CHaO.CieHsiO 

Palmitin,  or  tripalmitin,  C5iH9806  =  CHO.Ci6H3iO.     Of  the  two  solid 

CH2O.C16H31O 
varieties  of  fats,  palmitin  is  the  one  which  occurs  in  predominant  quan- 
tities in  human  fat  (Langer  i).  Palmitin  is  present  in  all  animal  fats  and 
in  several  kinds  of  vegetable  fat.  A  mixture  of  stearin  and  palmitin  was 
formerly  called  margarin.  As  to  the  occurrence  of  palmitic  acid,  C16H32O2, 
about  the  same  remarks  apply  as  to  stearic  acid.  The  mixture  of  these 
two  acids  has  been  called  margaric  acid,  and  this  mixture  occurs — often 
as  very  long,  thin,  crystalline  plates — in  old  pus,  in  expectorations  from 
gangrene  of  the  lungs,  etc. 

Palmitin  crystallizes,  on  cooling  from  a  warm  saturated  solution  in  ether 
or  alcohol,  in  starry  rosettes  of  fine  needles.  The  mixture  of  palmitin  and 
stearin,  called  margarin,  crystallizes,  on  cooling  from  a  solution,  as  balls  or 
round  masses  which  consist  of  short  or  long,  thin  plates  or  needles  which 
often  appear  like  blades  of  grass.  Palmitin,  like  stearin,  has  a  variable 
melting  and  solidifying  point,  depending  upon  the  way  it  has  been  pre- 
viously treated.  The  melting-point  is  often  given  as  62°  C.  According 
to  other  statements,^  it  melts  at  50.5°  C,  solidifies  on  further  heating, 
and  melts  again  at  66.5°  C. 
CH3 

Palmitic  acid,  (CH2)i4,  crystallizes  from  an  alcoholic  solution  in  tufts 
COOH 
of  fine  needles.  It  melts  at  62°  C;  still  the  admixture  mth  .stearic  acid, 
as  Heixtz  has  shown,  essentially  changes  the  melting-  and  solidify ing-points 
according  to  the  relative  amounts  of  the  two  acids.  Palmitic  acid  is  .some- 
what more  soluble  in  cold  alcohol  than  stearic  acid;  but  they  have  about 
the  same  solubility  in  boiling  alcohol,  ether,  chloroform,  and  benzene.  Its 
barium  salt  contains  21.17  per  cent  barium,  and  the  silver  salt  contains 
29.72  per  cent  silver. 

CH2O.C18H33O 

Olein,  or  triolein,  C57Hio406  =  CHO.Ci8H330,  is  present  in   all  animal 

CH2O.C18H33O 
fats,  and  in  greater  quantities  in  vegetable  fats.     It  is  a  solvent  for  stearin 
and  palmitin.     The  oleic  acid  (elaic  acid),  C18H34O2,  has  as  soaps  probably 
about  the  same  occurrence  as  the  other  fatty  acids. 

Olein  is,  at  ordinary  temperatures,  a  nearly  colorless  oil  of  a  specific 

'  Monat.shefte  f.  Chcm.,  2;    see  also  Jaeckle,  Zeitschr.  f.  phy.siol.  Chem.,  36. 
'  R.  Benedikt,  Analyse  der  Fette,  3.  Aufl.,  1897,  p.  44. 


OLEIC  ACID.  135 

gravity  of  0.914,  without  odor  or  marked  taste,  and  solidifies  in  cr}'staliine 
needles  at   —6°  C.     It  becomes  rancid  quickly  if  exposed  to  the  air.     It 
dissolves  with  difficulty  in  cold  alcohol,  but  more  easily  in  warm  alcohol 
or  in  ether.     It  is  converted  into  its  isomer,  elaidin,  bv  nitrous  acid. 
CHs 
(CHo)7 

/iTT 

Oleic  acid,   -„      ,  forms  on  heating,  besides  volatile  acids,  sebacic  acid, 

m-2)7 

COOH 
CioHi804,  which  crv'stallizes  in  shining  leaves  and  melts  at  127°  C.  With 
nitrous  acid  oleic  acid  is  transformed  into  the  isomeric  solid  elaidic  acid, 
which  melts  at  45°  C.  Oleic  acid  forms  at  ordmary-  temperature  a  colorless, 
tasteless,  and  odorless  oily  liciuid  which  solidifies  in  crystals  at  about  4°  C, 
which  then  melt  again  at  14°  C.  Oleic  acid  is  insoluble  in  water,  but  dis- 
solves in  alcohol,  ether,  and  chloroform.  With  concentrated  sulphuric 
acid  and  some  cane-sugar  it  gives  a  beautiful  red  or  reddish-violet  liquid 
whose  color  is  similar  to  that  produced  in  Pettenkofer's  test  for  bile- 
acids.  Oleic  acid  is  an  unsaturated  fatty  acid  which  can  take  up  halogens. 
Chi  heating  with  hydriodic  acid  and  amorphous  phosphorus  it  takes  up 
hydrogen  and  is  converted  into  stearic  acid.  Oleic  acid  readily  oxidizes 
in  the  air,  yieldmg  acid  products.  The  monoxystearic  acid  found  in 
certain  animal  fats  may  be  formed  from  oleic  acid  by  oxidation.  The 
barium  salt  of  oleic  acid  contains  19.65  per  cent  barium  and  the  silver  salt 
27.73  per  cent  silver. 

If  the  watery  solution  of  the  alkali  compounds  of  oleic  acid  is  pre- 
cipitated with  lead  acetate,  a  white,  tough,  sticky  mass  of  lead  oleate  is 
obtained  which  is  not  soluble  in  water  and  only  slightly  in  alcohol,  but  is 
soluble  in  ether.  This  salt  is  more  easily  soluble  in  benzene  than  the  lead 
salts  of  stearic  and  palmitic  acids,  and  this  behavior  of  the  lead  salts  towards 
ether  and  benzene  is  made  use  of  in  separating  oleic  acid  from  the  other 
fatty  acids. 

An  acid  related  to  oleic  acid,  doeglic  acid,  which  is  solid  at  0°  C,  liquid  at 
16°  C,  and  soluble  in  alcohol,  is  found  in  the  blubber  of  the  Baloena  rostrata. 
KrRBATOFF  '  has  demonstrated  the  presence  of  I'noleic  acid  in  the  fat  of  the  silurus, 
sturgeon,  seal,  and  certain  other  animals.  Drying  fats  have  also  been  found  by 
Amthor  and  Zink  ^  in  hares,  wild  rabbits,  wild  boar,  and  mountain-cock. 

To  detect  the  presence  of  fat  in  an  animal  fluid  or  tissue  the  fat  must 
first  be  shaken  out  or  extracted  with  ether.  After  the  evaporation  of  the 
ether  the  residue  is  tested  for  fat  and  the  acrolein  test  must  not  be  neg- 
lected. If  this  test  gives  positive  results,  then  neutral  fats  are  present; 
if  the  results  are  negative,  then  only  fatty  acids  are  present.     If  the  above 

»  Maly's  Jahresber.,  22.  '  Zeitschr.  f.  anal>^.  Chem.,  36. 


136  THE  ANIMAL  FATS. 

residue  after  evaporation  gives  the  acrolein  test,  then  a  small  portion  is 
dissolved  in  alcohol-ether  free  from  acid  and  which  has  been  colored  bluish 
violet  by  tincture  of  alkanet.  if  the  color  becomes  red,  a  mixture  of 
neutral  fat  and  fatty  acids  is  present.  In  this  case  the  fat  is  treated  while 
warm  with  a  soda  solution  and  evaporated  on  the  water-bath,  with  constant 
stirring  until  all  the  water  is  removed.  The  fatty  acids  hereby  combine 
with  the  alkali,  forming  soaps,  while  the  neutral  fats  are  not  saponihed 
under  these  conditions.  If  this  mixture  of  soaps  and  neutral  fats  is 
treated  with  water  and  then  shaken  with  pure  ether,  the  neutral  fats  are 
dissolved,  while  the  soaps  remain  in  the  watery  solution.  The  fatty  acids 
may  be  separated  from  this  solution  by  the  addition  of  a  mineral  acid 
which  sets  the  acid  free. 

The  neutral  fats  separated  from  the  soaps  by  means  of  ether  are  often 
contaminated  with  cholesterin,  which  must  be  separated  in  quantitative 
determinations  by  saponification  with  alcoholic  caustic  potash.  The 
cholesterin  is  not  attacked  by  the  caustic  alkali,  while  the  neutral  fats 
are  saponified.  After  the  evaporation  of  the  alcohol  the  residue  is  dissolved 
in  water  and  shaken  with  ether,  which  dissolves  the  cholesterin.  The  fatty 
acids  are  separated  from  the  watery  solution  of  the  soaps  by  the  addition 
of  a  mineral  acid.  If  a  mixture  of  soaps,  neutral  fats,  and  fatty  acids  is 
originally  present,  it  is  treated  first  with  water,  then  agitated  with  ether 
free  from  alcohol,  which  dissolves  the  fat  and  fatty  acids,  while  the  soaps 
remain  in  the  solution,  with  the  exception  of  a  very  small  amount  which  is 
dissolved  by  the  ether. 

To  detect  and  to  separate  the  different  varieties  of  neutral  fats  from 
each  other  it  is  best  first  to  saponify  them  with  alcoholic  potash,  or  still 
better  with  sodium  alcoholate,  according  to  Kossel,  Obermuller,  and 
Kruger.i  After  the  evaporation  of  the  alcohol  the  salts  of  the  fatty  acids 
are  dissolved  in  water  and  precipitated  with  sugar  of  lead.  The  lead  oleate 
is  then  separated  from  the  other  two  lead  salts  by  repeated  extraction  with 
ether,  but  it  must  be  remarked  that  the  lead  salts  of  the  other  fatty  acids 
are  not  quite  insolu])le  in  ether.  The  residue  insoluble  in  ether  is  decom- 
posed on  the  water-bath  with  an  excess  of  soda  solution,  evaporated  to 
dryness,  finely  pulverized,  and  extracted  with  boiling  alcohol.  The  alco- 
holic solution  is  then  fractionally  precipitated  by  barium  acetate  or  bariimi 
chloride.  In  one  fraction  the  amount  of  barium  is  determined,  and  in  the 
other  the  melting-point  of  the  fatty  acid  set  free  by  a  mineral  acid.  The 
fatty  acids  occurring  originally  in  the  animal  tissues  or  fluids  as  free  acids 
or  as  soaps  are  converted  into  barium  salts  and  investigated  as  above. 
According  to  Jaeckle,^  it  is  better  to  isolate  the  fatty  acids  as  silver  salts. 
This  same  experimenter  also  considers  it  more  advisable  to  dissolve  the  lead 
salts  in  warm  benzene,  as  suggested  by  Farnsteiner,  and  to  obtain  the 
crystalline  lead  salts  of  the  solid  fatty  acids  by  cooling. 

In  addition  to  the  methods  already  suggested  there  are  other  chemical  meth- 
ods which  are  important  in  investigating  fats.  Besides  ascertaining  the  melting- 
and  congealing-point  we  also  determine  the  following:  1.  The  acid  equivalent, 
which  is  a  measure  of  the  amount  of  fatty  acids  in  a  fat  and  is  determined  by 
titrating  the  fat  dissolved  in  alcohol-ether  with  N/10  alcoholic  caustic  potash, 
usmg  phenolphthalein  as  indicator.     2.  The  sajwnification  equivalent,  which  gives 

^  Zeitschr.  f.  physiol.  Chem.,  14,  15,  and  IQ. 
2  Ibid  .  36. 


INVESTIGATIOXS  OF  FATS.  137 

the  milligrams  of  caustic  potash  united  -with  the  fatty  acids  in  the  saponification 
of  1  gram  fat  with  X/2  alcoholic  caustic  potash.  3.  Reichert-Meissl's  equiva- 
lent, which  gives  the  quantity  of  volatile  fatty  acids  contained  in  a  given  amount 
of  neutral  fat  (5  grams).  The  fat  is  saponified,  then  acidified  with  mineral  acid 
and  distilled,  whereby  the  volatile  fatty  acids  pass  over  and  the  distillate  is 
titrated  with  alkali,  -i.  ludine  equivalent  is  the  cjuantity  of  iodine  absorbed  by  a 
certain  amount  of  the  fat  by  addition.  It  is  chieflj'  a  measure  of  the  quantity 
of  unsatm-ated  fatty  acids,  principally  oleic  acid  or  olein,  in  the  fat.  Other  bodies, 
such  as  cholesterin,  may  also  absorb  iodine  or  halogens.  The  iodine  equiva- 
lent is  generally  determined  according  to  the  method  suggested  by  v.  Hubl. 
5.  The  acetyl  equivalent.  Oxyacids,  alcohols  such  as  cetyl  alcohol  or  cholesterin, 
and  those  constituents  of  fats  containing  the  OH  group  are  transformed  into  the 
corresponding  acetyl  ester  on  boiling  v>'ith  acetic  anhydride,  while  the  fatty  acids 
remain  unchanged,  and  in  this  way  the  estimation  of  these  bodies  is  possible.  The 
fat  is  saponified,  the  soaps  decomposed  by  an  excess  of  acid,  and  the  mixture 
of  fatty  acids,  oxyfatty  acids,  cholesterin,  etc.,  boiled  with  acetic  anhydride. 
The  acid  equivalent  is  determined  in  a  weighed  part  of  the  carefully  washed 
acetic-acid-free  mixture  by  titration  with  alcoholic  caustic  potash.  This  acid 
equivalent  represents  all  the  acids  (fatty  acids  as  well  as  the  acetylated  oxyacids), 
and  it  is  designated  the  acetyl-acid  equivalent.  The  neutral  fluid  is  now  titrated 
with  an  exactly  measured,  sufficient  quantity  of  the  same  alkali  and  the  acetyl 
compounds  saponified  by  boiling.  On  retitrating  we  find  the  quantity  of  alkali 
used  in  saponification,  and  this  number,  calculated  to  100  parts  of  the  fat,  repre- 
sents the  acetyl  equivalent.  In  regard  to  the  performance  of  the  above-mentioned 
different  estimations  we  must  refer  the  reader  to  more  complete  works,  such  as 
"Analysis  of  Fats  and  Waxes,"  R.  Bexedikt,  1897. 

In  the  quantitative  estimation  of  fats  the  finely  divided  dried  tissues 
or  the  finely  divided  residue  from  an  evaporated  fluid  is  extracted  Anth 
ether,  alcohol-ether,  benzene,  or  any  other  proper  extraction  medium.  The 
investigations  of  Dormeyer  ^  and  others,  carried  on  in  PpLiJGER's  labora- 
toiy .  have  shouii  that  even  ^\-ith  veiy  prolonged  extraction  with  ether  all  the 
fat  is  not  extracted.  First  extract  the  greater  part  of  the  fat  b}^  ether. 
Then  digest  with  pepsin-hydrochloric  acid,  collect  the  insoluble  residue  on 
a  filter,  dr\',  and  extract  with  ether.  The  fat  is  extracted  from  the  filtrate 
by  shaking  uith  ether,  evaporatmg  the  extract  and  the  fat  separated  from 
other  bodies  by  extracting  the  residue  with  petroleum  ether.  Lecitliui 
and  other  bodies  are  dissolved  by  the  various  solvents,  hence  the  results 
for  the  fats  may  be  too  high.  This  is  especially  the  case  on  using  the  saponi- 
fication method  -  suggested  by  Liebermaxx  and  Szekely,  whereby  the  leci- 
thins as  well  as  the  fats  are  saponified.  Glikix  3  recommends  as  the  best 
procedure  the  extraction  with  boiling  petroleum  ether  and  the  removal  of 
the  lecithin  by  acetone,  in  which  it  is  insoluble. 

The  fats  are  poor  in  oxygen,  but  rich  in  carbon  and  hydrogen.  They 
therefore  represent  a  large  amount  of  chemical  potential  energy,  and  yield 
correspondingly  large  quantities  of  heat  on  combustion.     The}^  take  first 

^  On  fat  extraction  for  quantitative  estimation  see  Dormeyer,  Pfliiger's  Arch.,  61 
and  65;  Bogdanow,  ibid.,  65,  6S,  and  Arch.  f.  (Anat.  u.)  physiol.,  1897,  149;  N.  Schulz, 
Pfliiger's  Arch.,  66;  Voit  and  Krummacher,  Zeitschr.  f.  Biologie,  35;  O.  Frank,  ibid., 
35,    Polimanti,  Pfliiger's  Arch.,  70;    J.  Nerking,  ibid.,  71. 

^  Pfliiger  s  Arch.,  72,  and  Liebermann,  ibid.,  lOS. 

''  Ibid. ,  95. 


138  THE   AXIMAL   FATS. 

rank  among  the  foods  in  this  regard,  and  are  therefore  of  \evy  great 
importance  in  animal  life.  We  will  speak  more  in  detail  of  this  signifi- 
cance, also  of  fat  formation  and  of  the  beha\'ior  of  the  fats  m  the  body,  in 
the  following  chapters. 

The  LECITHINS,  which  stand  in  close  relationship  to  the  fats,  will  be 
treated  in  a  subsequent  chapter  (Y).  The  following  bodies  are  related 
to  the  ordinary-  animal  fats. 

Spermaceti.  In  the  living  spermaceti  or  white  whale  there  is  found  in  a  large 
cavity  in  the  skull  an  oily  liquid  called  spermaceti,  which  on  cooling  after  death 
separates  into  a  solid  crystalline  part  ordinarily  called  spermaceti,  and  into  a 
liquid,  SPERMACETI-OIL.  This  last  is  separated  by  pressure.  Spermaceti  is  also 
found  in  other  whales  and  in  certain  species  of  dolphin. 

The  purified,  solid  spermaceti,  which  is  called  cetin,  is  a  mixture  of  esters  of 
fatty  acids.  The  chief  constituent  is  the  cetyl-palmitic  ester  mixed  with  small 
quantities  of  compound  esters  of  lauric,  myristic,  and  stearic  acids  with  radicals 
of  the  alcohols,  lethal,  CjHas.OH,  methal,  C,4H29.0H,  and  stethal,  CisH3;.0K. 

Cetin  is  a  snow-white  mass  shining  like  mother-of-pearl,  crystallizing  in  plates, 
brittle,  fatty  to  the  touch,  and  which  has  a  varying  melting-point  of  30°  to 
50°  C,  depending  upon  its  purity.  Cetin  is  insoluble  in  w^ater,  but  dissolves 
easily  in  cold  ether  or  volatile  and  fatty  oils.  It  dissolves  in  boiling  alcohol, 
but  crystallizes  on  cooling.  It  is  saponified  with  diSiculty  by  a  solution  of  caustic 
potash  in  water,  but  with  an  alcoholic  solution  it  saponifies  readily  and  the  above- 
mentioned  alcohols  are  set  free. 

CH3 

Ethal  or  cetyl  alcohol,  CeHg^O  =  (CH2)i4,  which  occurs  in  smaller  quar  titles 

CH2.OH 
in  beeswax,  and  was  found  by  Ludwig  and  v.  Zeynek  '  in  the  fat  from  dermoid 
cysts,  forms  white,  transparent,  odorless,  and  tasteless  crystals  which  are  insoluble 
in  water  but  dissolve  easily  in  alcohol  and  ether.     Ethal  melts  at  49.5°  C. 

Spermaceti-oil  yields  on  saponification  valerianic  acid,  small  amounts  of 
solid  fatty  acids,  and  physetoleic  acid.  This  acid,  which  has,  like  hypogaeic 
acid,  the  composition  CibH»)02,  occurs  also,  as  found  by  Ljubarsky,^  in  con- 
siderable amounts  in  the  fat  of  the  seal.  It  forms  colorless  and  odorless  needle- 
shaped  crystals  which  easily  dissolve  in  alcohol  and  ether  and  melt  at  34°  C. 

Beeswax  may  be  treated  here  as  concluding  the  subject  of  fats.  It  con- 
tains three  chief  constituents:  (1)  cerotic  acid,  CjeHsjOj,^  which  occurs  as  cetyl 
ether  in  Chinese  wax  and  as  free  acid  in  ordinary  w^ax.  It  dissolves  in  boiling 
alcohol  and  separates  as  crystals  on  cooling.  The  cooled  alcoholic  extract  of 
wax  contains  (2)  cerolein,  which  is  probably  a  mixture  of  several  bodies,  and 
(3)  MYRiciN,  which  forms  the  chief  constituent  of  that  part  of  w-ax  which  is  in- 
soluble in  warm  or  cold  alcohol.  Myricin  consists  chiefly  of  palmitic-acid  ester 
of  melissji  (myricyl)  alcohol,  C^  Hgi.OH.  This  alcohol  is  a  silky,  shining,  crys- 
talline body  melting  at  85°  C. 

'  Zeitschr.  f.  physiol.  Chem.,  23. 

2  Journ.  f.  prakt.  Chem.  (N.  F.),  57. 

'See  Henriques,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30,  1415. 


CHAPTER  V. 
THE  ANIMAL  CELL. 

The  cell  is  the  unit  of  the  manifold  variable  forms  of  the  organism;  it 
forms  the  simplest  physiological  apparatus,  and  as  such  is  the  seat  of 
chemical  processes.  It  is  generally  admitted  that  all  chemical  processes 
of  importance  do  not  take  place  in  the  animal  fluids,  but  transpire  in  the 
cells,  hence  the  cell  may  be  considered  as  the  chemical  laboratory  of  the 
organism.  It  is  also  principally  the  cells  wliich,  through  their  greater  or 
less  activity,  regulate  or  govern  the  range  of  the  chemical  processes,  and 
also  the  extensiveness  of  the  total  exchange  of  material. 

It  is  natural  that  the  chemical  investigation  of  the  animal  cell  should 
in  most  cases  be  in  reality  a  study  of  those  tissues  of  which  it  forms 
the  chief  constituent.  Only  in  a  few  cases  can  the  cells,  by  relatively 
simple  manipulations,  be  directly  isolated  in  a  rather  pure  state  from  the 
tissues,  as,  for  example,  in  the  investigation  of  pus  or  of  tissues  very  rich 
in  cells.  But  even  in  these  cases  the  chemical  investigation  may  not  lead 
to  any  positive  results  in  regard  to  the  constituents  of  the  uninjured  living 
cells.  By  the  process  of  chemical  transformation  new  substances  may  be 
formed  on  the  death  of  the  cell,  and  at  the  same  time  physiological  con- 
stituents of  the  cell  may  be  destroyed  or  transported  into  the  surrounding 
medium  and  therefore  escape  investigation.  For  this  and  other  reasons 
we  possess  only  a  veiy  limited  knowledge  of  the  constituents  and  the  com- 
position of  the  cell,  especially  of  the  living  one. 

While  young  cells  of  different  origin  in  the  early  period  of  their  exist- 
ence may  show  a  certain  similarity  in  regard  to  form  and  chemical  com- 
position, they  may,  on  further  development,  not  only  take  the  most  varied 
forms,  but  may  also  offer  from  a  chemical  standpoint  the  greatest  diversity. 
As  a  description  of  the  constituents  and  composition  of  the  different  cells 
occurring  in  the  animal  organism  is  nearly  equivalent  to  a  demonstratioQ 
of  the  chemical  properties  of  most  animal  tissues,  and  as  this  exposition 
will  be  found  in  the  corresponding  chapters,  we  ^^dll  here  discuss  only  the 
chemical  constituents  of  the  young  cells  or  cells  in  general. 

In  the  study  of  these  const  Huerts  we  are  confronted  with  another 
difficulty,  namely,  we  must   different  iate  by  chemical  research  between 

139 


140  THE   ANIMAL  CELL. 

those  constituents  which  are  essentially  necessar}-  for  the  life  of  the  cells 
and  those  which  are  casual,  i.e.,  stored  up  as  reserve  material  or  as  meta- 
bolic products.  In  tliis  connection  we  have  only  been  able,  thus  far,  to 
learn  of  certain  substances  wliich  seem  to  occur  in  every  developing  cell. 
Such  bodies,  called  pri^l\ry  by  Kossel,i  are,  besides  water  and  certain 
mineral  constituents,  proteins,  nucleoproteids  or  nucleins,  lecitliins,  gly- 
cogen (?),  and  cholesterin.  Those  bodies  which  do  not  occur  in  every 
developing  cell  are  called  secondary.  Among  these  we  have  fat,  gly- 
cogen (?),  pigments,  etc.  It  must  not  be  forgotten  that  it  is  still  possible 
that  other  primar}-  cell  constituents  may  exist,  as  yet  unkno\\'n  to  us,  and 
we  also  do  not  know  whether  all  the  primary  constituents  of  the  cell  are 
necessary'  or  essential  for  the  life  and  functions  of  the  same. 

,\nother  important  question  is  the  division  of  the  various  cell  constit- 
uents between  the  two  morphological  components  of  the  cell,  namely,  the 
protoplasm  and  the  nucleus.  This  is  very  difficult  to  decide  for  many  of 
the  constituents;  ne^■ertheless  it  is  appropriate  to  differentiate  between 
the  protoplasm  and  the  nucleus. 

The  Protoplasm  of  the  developing  cell  consists  during  life  of  a  semi- 
solid mass,  contractile  under  certain  conditions  and  readily  changeable, 
which  is  rich  in  water  and  whose  cliief  portion  consists  of  protein  sub- 
stances, i.e.,  of  colloids.  If  the  cell  be  deprived  of  the  physiological  con- 
ditions of  life,  or  if  exposed  to  destmctive  exterior  influences,  such  as  the 
action  of  high  temperatures  or  of  chemical  agents,  the  protoplasm  dies.  The 
protein  bodies  which  it  contains  coagulate  at  least  partially,  and  other 
chemical  changes  are  found  to  take  place.  The  alkaline  reaction  (litmus) 
of  the  living  cell  may  become  acid  by  the  appearance  of  paralactic  acid, 
and  the  carbohydrate,  glycogen,  which  habitually  occurs  in  manj^  cells, 
may  after  their  death  be  quickly  changed  and  consumed. 

The  question  as  to  the  internal  structure  of  the  protoplasm  is  still  in 
controversy.  It  is  of  little  importance  in  the  study  of  the  chemical  com- 
position of  the  cells,  as  it  is  impossible  to  study,  especially  by  chemical 
means,  the  morphologically  different  constituents  of  the  protoplasm.  With 
the  exception  of  a  few  microchemical  reactions  the  chemical  analysis  has 
been  restricted  to  the  protoplasm  as  such,  and  the  investigations  have 
been  directed  in  the  first  place  to  the  protein  substances  which  form  the 
chief  mass  of  the  protoplasm. 

The  proteins  of  the  protoplasm  consist,  according  to  the  older  general 
view,  chiefly  of  globulins.  Albumins  have  also  been  found  besides  the 
globulins.  There  is  no  doubt  at  present  that  the  albumins  occur  in  the 
Cells  only  as  traces,  or  at  least  only  in  trifling  quantities.  The  presence  of 
globulins  can  hardly  be  disputed,  although  certain  cell   constituents  de- 

*  Verhandl.  d.  physiol.  Geselisch.  zu  Berlin,  1S90-91.  Nos.  5  and  0. 


PROTEINS  OF  THE  CELL.  141 

scribed  as  globulins  have  been  sho^\^l  on  closer  investigation  to  be 
nucleoalbumins  or  nucleoproteids.  According  to  Halliburton  ^  the 
protein  occurring  in  all  cells  and  coagulating  at  47-50°  C.  is  a  true 
globulin. 

In  opposition  to  the  ^dew  that  the  chief  mass  of  the  animal  cell  consists 
of  true  proteicls,  Hammarstex^  expressed  the  opinion  several  years  ago 
that  the  chief  mass  of  the  protein  substances  of  the  cells  does  not  consist 
of  proteids  in  the  ordmary  sense,  but  consists  of  more  complex  phosphor- 
ized  bodies,  and  that  the  globulins  and  albumms  are  to  be  considered  as 
nutritive  material  for  the  cells  or  as  destructive  products  in  the  chemical 
transformation  of  the  protoplasm.  This  view  has  received  substantial 
support  by  investigations  within  the  last  few  years.  Alex.  Schmidt^  has 
come  to  the  \'iew,  by  investigations  on  various  kinds  of  cells,  that  they 
contain  only  ver\'  little  proteid,  and  that  the  chief  mass  consists  of  very 
complex  protein  substances. 

The  protein  substances  of  the  cells  consist  chiefly  of  compound  proteids, 
and  these  are  divided  between  the  glucoproteid  and  the  nucleoproteid 
groups.  It  is  impossible  at  present  to  state  to  what  extent  nucleoalbumuis 
exist  in  the  cells,  because  thus  far  in  most  cases  no  exact  difference  has  been 
made  between  them  and  the  nucleoproteids.  Hoppe-Seyler  "*  calls  vitelUn 
a  regular  constituent  of  all  protoplasm.  This  body  used  to  be  considered 
as  a  globulin,  but  later  researches  have  sho\Mi  that  the  so-called  \'itellin 
bodies  may  be  of  various  kinds.  Certain  \'itellins  seem  to  be  nucleo- 
albimiins,  and  it  is  therefore  \ery  probable  that  cells  habitually  contain 
nucleoalbumins. 

The  nucleoproteids  take  a  verj-  prominent  place  among  the  compound 
proteids  of  the  cell.  The  various  substances  isolated  by  different  investiga- 
tors from  animal  cells,  such  as  tissue-fibrinogen  (Wooldridge),  cytoglobin 
and  prdglobulin  (Alex.  Schmidt),  or  nucleohistone  (Kossel  and  Liliex- 
FELD^),  belong  to  this  group.  The  cell  constituent  which  swells  up  to  a 
sticky  mass  ^\ith  common  salt  solution  and  is  called  Ro^^DA's  hyaline  sub- 
stance  also  belongs  to  this  group. 

The  above-mentioned  different  protein  substances  have  simply  been 
designated  as  constituents  of  the  cells.  The  next  question  is  which  of 
these  belong  to  the  protoplasm  and  which  to  the  nucleus.  At  present 
we  can  give  no  positive  answer  to  this  question.     According  to  Kossel 

'  See  Halliburton,  On  the  Chemical  Physiology  of  the  Animal  Cell,  1893,  No.  1, 
King's  College  Physiol.  Laboratory. 

2  Pfliiger's  Arch.,  36,  449. 

3  Alex.  Schmidt,  Zur  Blutlehre,  Leipzig,  1892. 
^Physiol.  Chem.,  1877-1881,  76. 

*  See  L.  C.  Wooldridge,  Die  Gerinnung  des  Blutes,  Leipzig,  1891;  A.  Schmidt, 
Zur  Blutlehre;    Lilienfeld,  Zeitschr.  f.  physiol.  Chem.,  18. 


142  THE  ANIMAL  CELL. 

and  LiLiENFELD,^  the  cell-nucleus  of  the  leucocytes  of  the  thymus  gland 
contains  a  nucleoproteid  as  chief  constituent,  besides  nucleins,  and  some- 
times perhaps  also  nucleic  acid  (see  below),  while  the  body  of  the  cells 
contains  chiefly  pure  proteids,  besides  other  substances,  and  a  nucleo- 
proteid, containing  only  a  very  small  quantity  of  phosphorus.  As  the 
lymphocytes  of  the  thymus  gland  of  the  calf  contain  only  one  nucleus,  in 
which  the  mass  of  the  nucleus  surpasses  that  of  the  cytoplasm,  it  is  natural 
that  the  relative  proportion  of  the  various  protein  substances  in  these  cells 
cannot  be  taken  as  a  standard  for  the  composition  of  other  cells  richer  in 
cytoplasm. 

Complete  investigations  in  regard  to  the  distribution  of  protein  sub- 
stances in  the  protoplasm  and  nucleus  of  other  cells  have  not  been  made. 
If  we  consider  for  the  present  that  the  cells  rich  in  protoplasm  contain,  as 
a  rule,  only  ver}'  little  true  proteid,  we  are  hardly  wrong  in  considering  it 
probable  that  the  protoplasm  contains  chiefly  nucleoalbumins  and  com- 
pound proteids  besides  traces  of  albumin  and  a  little  globulin.  These  com- 
pound proteids  are  in  certain  cases  glucoproteids,  but  otherwise  nucleo- 
proteids,  which  differ  from  the  nucleoproteids  of  the  nucleus  in  being 
poorer  in  phosphorus,  besides  containing  a  great  deal  of  proteid  and  only 
a  little  of  the  prosthetic  group,  and  hence  have  no  specially  pronounced 
acid  character. 

The  nucleoproteids  of  the  nucleus  are  on  the  contrar}^,  as  shown  by 
LiLiENFELD  and  KossEL,  rich  in  phosphorus  and  of  a  strongly  acid  charac- 
ter. These  nucleoproteids  will  be  treated  in  speaking  of  the  nucleic  acids 
of  the  nucleus. 

In  cases  in  which  the  protoplasm  is  surrounded  by  an  outer,  condensed 
layer  or  a  cell  membrane,  this  envelope  seems  to  consist  of  albuminoid 
substances.  In  a  few  cases  these  substances  seem  to  be  closely  related  to 
elastin;  in  other  cases,  on  the  contrary,  they  seem  rather  to  belong  to  the 
keratin  group.  Even  in  cells  w-hich  do  not  seem  to  have  any  visible  special 
layers  forming  boundaries,  we  still  admit  of  such  layers  on  account  of  the 
behavior  of  the  cells  as  regards  permeability. 

Nernst^  has  shown  by  a  special  experiment  that  the  permeability  of  a 
membrane  for  a  certain  substance  is  essentially  dependent  upon  the  sol- 
vent power  of  the  membrane  for  the  said  substance.  This  point,  which 
is  of  the  greatest  importance  in  the  study  of  osmotic  phenomena  in  living 
cells,  has  been  specially  investigated  by  Overton.^  The  behavior  of  the 
living    cells  towards  dyestuffs,   also  the  ready  introduction  into  animal 

'  Ueber  die  Wahlverwandtschaft  der  Zellelemente  zu  gewissen  Farbstoffen,  Ver- 
handl.  d.  physiol.  Gesellsch.  zu  Berlin,  No.  11,  1893. 

^  Zeitschr.   f.  physikal.  Chem.,  6. 

'  Vierteljahrsschr.  d.  Naturf.  Ges.  in  Zurich,  44  (1899),  and  Overton,  Studien  iiber 
die  Narkose,  Jena,  1901. 


LECITHLXS.  143 

and  plant  protoplasm  of  such  bodies  as  are  insoluble  or  only  slightly 
soluble  in  water  but  readily  soluble  in  fats  or  fat-like  bodies,  has  led 
Overton  to  conclude  that  the  protoplasm-boundary  layer  behaves  like  a 
substance  layer  whose  solvent  power  is  closely  related  to  the  fatty  oils. 
According  to  this  investigator,  the  protoplasm-boundary  layer  is  probably 
impregnated  with  lipoids,  i.e.,  with  lecithins,  cholesterin,  and  bodies  similar 
to  protagon,  and  among  which  lecithin,  which  also  takes  up  water,  must 
be  of  the  greatest  importance. 

The  cholesterins  and  the  protagons  will  be  best  treated  in  another 
connection  (see  Chapters  VIII  and  XII).  We  will  discuss  here  only  the 
lecithins,  which  are  present  in  every  cell. 

Lecithins.  These  bodies  are  ester  compounds  ^  of  glycerophosphoric 
acid  substituted  b}'  two  fatty-acid  radicals  with  a  base  called  choline. 
According  to  the  kind  of  fatty  acid  contained  in  the  lecithin  molecule  it 
is  possible  to  have  various  lecithins,  such  as  stear}'l-,  palmityl-,  and  oleyl- 
lecithins.  According  to  Thudichum  ^  two  different  fatty  acids  may  exist 
simultaneously  in  one  lecithin,  and  according  to  him  ever}-  true  lecithin 
always  contains  at  least  one  oleic-acid  radical.  All  lecithins  are  mono- 
nitrogenous  monophosphatides,  which  contain  1  atom  of  nitrogen  for 
every  atom  of  phosphorus.  As  an  example  of  a  lecithin  we  give  the  ore 
closely  studied  by  Hoppe-Seyler  and  Diaconow,^  called  distearyl-lecithin, 

CH2 — O — C18H35O 

I 
CH-  O-C18H35O 

C44H9oNP09=  CH,— 0\ 

HO^PO. 
/C,H4— 0/ 

Nf(CH3)3 
\0H 

According  to  Henriques  and  Hansen ^  the  iodine  equivalent  of  the 
fluid  fatty  acids  obtained  from  egg  as  well  as  brain  lecithin  is  higher  than 
that  of  oleic  acid,  hence  it  follows  that  the  lecithins  contain  other  fatty 
acids  besides  stearic,  palmitic,  and  oleic  acids. 

Erlandsen  ^  in  a  specially  thorough  ard  careful  investigation  has  studied 
the  phosphatides  of  the  ox  heart  and  ox  muscles.  The  lecithin  had  the 
same  composition  as  that  from  the  egg-yolk.     The  iodine  equivalent  as 

*  Streoker,  Annal.  d.  Chem.  u.  Fharm.,  148;  Hundeshagen,  Journ.  f.  prakt.  Chem. 
(N.  F.),  28;    Gilson,  Zeitschr.  f.  physiol.  Chem.,  12. 

^  J.  L.  W.  Thudichum,  Die  chemische  Konstitution  des  Gehirns  des  Menschen,  etc., 
Tubingen,  1901. 

^Hoppe-Seyler,  Med.  chem.  I'ntersuch.,  Heft  2  and  3. 

*  Skand.  Arch.  f.  Physiol.,  14. 

^A.  W.  E.  Erlandsen,  Undersogelser  over  Hjertets  Phosphatider,  Copenhagen,  1906- 


144  THE  ANIMAL  CELL. 

well  as  the  analysis  show  that  the  fatty  acids  occurring  in  the  lecithin  mole- 
cule are  very  poor  in  hydrogen  and  belong  in  part  to  the  linolic  or  linolenic 
acid  series.  Diaminomonophosphatides,  i.e.,  compounds  in  which  the 
relationship  N:P  is  not,  as  in  lecithin,  1:1,  but  2:1,  occur  in  the  muscles, 
but  chiefly  in  the  heart  muscle.  These  phosphatides  are  isolated  as  metal- 
lie  salts,  and  the  cadmium  compound  of  the  diaminomonophosphatide 
obtained  from  the  heart  had  the  composition  C4oH75N2POi2-2CdCl2. 
Erlandsen  has  isolated  a  new  phosphatide  from  the  heart,  which  he 
calls  cuorin  and  which  belongs  to  the  group  of  monaminodiphosphatides, 
in  which  the  relation  of  N:P  is  1:2.  This  cuorin,  which  occurs  only  in 
traces  in  other  muscles,  contains  two  phosphoric-acid  radicals  which  in 
part  are  united  with  glyceryl.  Besides  these  it  contains  two  residues  of 
strongly  unsaturated  fatty  acids  and  a  basic  radical,  which  is  not  identical 
with  choline.  The  empirical  formula  is  C7iHi25NP202i.  Cuorin  is  soluble 
in  ether  but  insoluble  in  alcohol,  and  is  characterized  by  a  very  great  auto- 
oxidizability.  It  is  obtained  in  the  amorphous  state.  The  monaminophos- 
phatides  (lecithin  and  cuorin)  can  be  directly  extracted  from  the  air-dried 
and  finely  dinded  organs,  and  to  all  appearances  occur  in  the  free  state. 
The  diaminophosphatides  are  also  soluble  in  ether,  but  cannot  be  directly 
extracted  by  ether,  but  only  after  a  previous  treatment  with  alcohol,  and 
therefore  probably  exist  in  combination  with  proteins. 

WiNTERSTEiN  and  HiESTAND,!  and  previous  to  them  Schulze  and  WiN- 
TERSTEiN,  have  isolated  from  different  parts  of  plants,  lecithin  preparations 
which  are  poorer  in  phosphorus  than  the  ordinary  lecithin,  containing  as  a 
maximum  2.74  per  cent  phosphoms,  and  which  on  cleavage  with  dilute 
mineral  acids  yielded,  besides  fatty  acids,  glycerophosphoric  acid,  and 
cholme,  also  considerable  quantities  of  hexoses,  indeed  16  per  cent.  The 
hexoses  were  cZ-glucose  and  c?-galactose,  and  besides  these  small  quantities 
of  pentoses  were  found.  These  phosphatides  seem  to  be  widely  distributed 
in  the  plant  kingdom. 

On  saponification  with  alkalies  or  baryta-water,  lecithin  yields  fatty 
acids,  glycerophosphoric  acid,  and  choline.  It  is  only  slowly  decomposed 
by  dilute  acids.  Besides  small  quantities  of  glycerophosphoric  acid  we 
have  large  quantities  of  free  phosphoric  acid  split  off. 

CH^.OH 

Glycerophosphoric    acid,  CgHgPOg^CH.OH  ,  is  a  bibasic  acid  which  prob- 

I 
CH-0\ 

oh4po 

OH/ 
ably  occurs  in  the  animal  fluids  and  tissues  only  as  a  cleavage  product  of  lecithins. 
According  to  Willstatter  and  Ludecke  ^  the  glycerophosphoric  acid   split  off 

*  Zeitschr.  f.  physiol.  Chem.,  47. 

^Willstatter  and  Ludecke,  Ber.  d.  d.  chem.  Gcsellsch.,  37. 


LECITHINS.  145 

from  lecithins  is  optically  active.  Its  barium  and  potassium  salts  are  levorota- 
tory  and  behave  in  certain  regards  differently  from  the  corresponding  salts  of 
synthetically  prepared  glycerophosphoric  acid. 

/CH,.CH,(OH) 
Choline  (trimethyloxyethylammoniumhydroxide),C5H,5N02  =  N— (0113)3  , 

\0H 
which  occurs  extensively  in  the  plant  kingdom,  is  not  identical  with  the  base, 
NEURiNE,  prepared  by  Liebreich  as  a  decomposition  product  from  the  brain, 
which  is  considered  as  trimethylvinylammonium  hydroxide,  C5H13NO.  Choline 
is  a  syrupy  fluid  readily  miscible  with  absolute  alcohol.  Hydrochloric  acid  gives  a 
compound  which  is  very  soluble  in  water  and  alcohol,  but  insoluble  in  ether, 
chloroform,  and  benzene.  This  compound  forms  a  double  combination  with  plati- 
num chloride  which  is  soluble  in  water,  insoluble  in  absolute  alcohol  and  ether, 
crystallizing  ordinarily  in  six-sided  orange-colored  plates.  This  compound  is 
used  in  the  detection  and  identification  of  this  base.  Choline  also  forms  a 
crystalline  double  compound  with  mercuric  chloride  and  with  gold  chloride. 
Choline  is  precipitated  by  potassium  iodide  and  iodine  (Gulewitsch),  and  potas- 
sium triiodide  can  be  used  for  the  ciuantitative  estimation  of  this  base  (Stanek  ^) . 
On  heating  the  free  base  it  decomposes  into  trimethylamine,  ethylene  oxide,  and 
water. 

Lecithin  occurs,  as  Hoppe-Seyler  2  has  especially  shown,  widely  diffused 
in  the  vegetable  and  animal  kingdoms.  According  to  this  investigator  it 
occurs  also  in  many  cases  in  loose  combination  with  other  bodies,  such  as 
proteins,  haemoglobin,  and  others.  Lecithin,  according  to  Hoppe-Seyler, 
is  found  in  nearly  all  animal  and  vegetable  cells  thus  far  studied,  and  also 
in  nearly  all  animal  fluids.  It  is  especially  abundant  in  the  brain,  nerves, 
fish  eggs,  yolk  of  the  egg,  electrical  organs  of  the  Torpedo  electricus,  semen, 
and  pus,  and  also  in  the  muscles  and  blood-corpuscles,  blood-plasma,  lymph, 
milk,  especially  woman's  milk,  and  bile.  Lecithin  is  also  found  in  differ- 
ent pathological  tissues  or  liquids. 

SiWERTZow^  has  determined  the  amount  of  lecithin  in  the  human 
foetus  and  in  children  of  various  ages,  and  he  finds  that  the  quantity  of 
lecitliin  is  much  greater  in  the  organs  (brain,  liver,  heart,  and  muscles) 
of  the  ripe  foetus  as  compared  with  the  same  organs  of  children  up  to  ten 
years  of  age.  The  child  according  to  him  has  a  certain  store  of  lecithin 
when  it  comes  into  the  world  and  this  is  consumed  during  the  first  months 
of  its  extra-uterine  life. 

This  wide  distribution  of  the  lecithins,  as  also  the  fact  that  they  are 
primary  cell  constituents,  gives  great  physiological  importance  to  these 
substances.  We  have  in  lecithin,  no  doubt,  a  verj-  important  material 
for  the  building  up  of  the  complicated  phosphorized  nuclein  substances  of 
the  cell  and  cell  nucleus.  That  the  lecithins  are  of  great  importance  in 
the  development  and  growth  of  li^dng  organisms,  in  fact  for  the  bioplast ic 

*  In  regard  to  choline  and  its  compounds  see  Gulewitsch,  Zeitschr.  f.  physiol, 
Chem.,  24;    Stanek,  ibicL,  -46. 

=>  Physiol.  Chemie,  Berlin,  1877-1881,  57.' 
^  See  Biochem.  Centralbl.,  2,  310. 


146  THE  ANIMAL  CELL. 

processes  in  general,  follows  also  from  several  investigations.^  The  fact 
must  not  be  overlooked  that  in  the  animal  body  we  find  besides  the  leci- 
thins also  other  related  phosphatides  which  have  been  little  studied  and 
which  can  be  readily  mistaken  for  lecithins. 

Lecithin  may  be  obtained  in  grains  or  warty  masses  composed  of  small 
crystalline  plates  by  strongly  cooling  its  solution  in  strong  alcohol.  In  the 
dry  state  it  has  a  waxy  appearance,  is  plastic,  but  forms  pulverizable  masses 
when  dried  in  vacuum,  and  is  soluble  in  alcohol,  especially  on  heating  (to 
40-50°  C);  it  is  less  soluble  in  ether.  It  is  dissolved  also  by  chloroform, 
carbon  disulphide,  benzene,  and  fatty  oils.  The  solution  of  lecithin  from 
egg-yolk  is  dextrorotatory  (Ulpiani^).  The  solution  of  lecithin  in  alcohol- 
ether  or  chloroform  is  precipitated  by  acetone.  It  swells  in  water  to  a 
pasty  mass  which  shows  under  the  microscope  slimy,  oily  drops  and  threads, 
so-called  myelin  forms  (see  Chapter  XII).  On  warming  this  swollen  mass 
or  the  concentrated  alcoholic  solution,  decomposition  takes  place  with 
the  production  of  a  brown  color.  On  allowing  the  solution  or  the 
swollen  mass  to  stand,  decomposition  takes  place  and  the  reaction  becomes 
acid. 

With  considerable  water,  lecithins  give  an  emulsion  or  indeed  a  filter- 
able colloidal  solution,  which  is  precipitated  by  salts  with,  divalent  cations, 
such  as  Ca,  ^Ig,  and  others  (W.  Koch).  This  precipitate  dissolves  again 
in  water  after  the  removal  from  the  solution  of  the  electrolytes,  and  the 
formation  of  this  precipitate  can  be  prevented  by  the  presence  of  salts  of 
monovalent  cations.  We  are  here  not  dealing  with  a  chemical  but  rather 
with  a  physical  precipitation  reaction  (Koch  3).  In  putrefaction  lecithins 
yield  glycerophosphoric  acid  and  choline;  the  latter  further  decomposes 
with  the  formation  of  methylamine,  ammonia,  carbon  dioxide,  and  marsh- 
gas  (Hasebroek^).  If  dry  lecithin  be  heated  it  decomposes,  takes  fire, 
and  bums,  leaving  a  phosphorized  ash.  On  fusing  with  caustic  alkali  and 
saltpetre  it  yields  alkali  phosphates.  Lecithins  are  easily  carried  down  dur- 
ing the  precipitation  of  other  compounds  such  as  the  protein  bodies,  and 
may  therefore  very  greatly  change  the  solubilities  of  the  latter. 

Lecithins  combine  with  acids  and  bases.  The  compound  with  hydro- 
chloric acid  give  with  platinum  chloride  a  double  salt  which  is  insoluble 
in  alcohol,  soluble  in  ether,  and  which  contains  10.2  per  cent  platinum 
(for  distearyl-lecithin).     The  cadmium-chloride  compound  which  contains 

'See  Stoklasa,  Ber.  d.  deutsch.  chem.  Gesellsch.,  29;  Wiener  Sitzungsber. ,  104; 
Zeitschr.  f,  physiol.  Chem.,  2.);  W.  Danilewsky,  Comp.  rend.,  121  and  123,  and  W. 
Koch,  Zeitschr.  f.  physiol.  Chem.,  3";  P.  Kyes,  ibid.,  41,  and  Berl.  klin.  Wochenschr., 
1904. 

2  Chem.  Centralbl.,  1901,  2,  30  and   193. 

^Zeitschr.  f.  physiol.  Chem.,  37. 

'Ibid.,  12. 


LECITHINS.  147 

3  molecules  of  lecithin  and  4  molecules  of  cadmium  chloride  (Ulpiam  i) 
is  difficultly  soluble  in  alcohol,  but  dissolves  in  a  mixture  of  carbon  disul- 
phido  and  ether  or  alcohol.  A  solution  of  lecithins  in  alcohol  is  not  pre- 
cipitated by  lead  acetate  and  ammonia. 

Lecithin  may  be  prepared  tolerably  pure  from  the  3'olk  of  the  hen's  egg 
by  the  following  methods,  as  suggested  by  Hoppe-Seyler  and  Diacoxow. 
The  yolk,  deprived  of  protein,  is  extracted  with  cold  ether  until  all  the 
yellow  color  is  removed.  Then  the  residue  is  extracted  with  alcohol  at 
50-60°  C.  After  the  evaporation  of  the  alcoholic  extract  at  50-60°  C,  the 
syrupy  matter  is  treated  with  ether  and  the  insoluble  residue  dissolved  in 
as  little  alcohol  as  possible.  On  cooling  this  filtered  alcoholic  solution  to 
—  5°  to  —10°  C.  the  lecithin  gradually  separates  in  small  granules.  The 
ether,  however,  contains  considerable  of  the  lecithin.  The  ether  is  dis- 
tilled off  and  the  residue  dissolved  in  chloroform  and  the  lecithin  precipi- 
tated from  this  solution  by  means  of  acetone  (Altmann). 

According  to  Gilson  ^  a  new  portion  of  lecithin  may  be  obtained  from 
the  ether  used  in  extracting  the  yolk  by  dissolving  the  residue  after  the 
evaporation  of  the  ether  in  petroleum-ether  and  then  shaking  this  solution 
with  alcohol.  The  petroleum-ether  takes  the  fat,  while  the  lecithin  re- 
mains dissolved  in  the  alcohol  and  may  be  obtained  therefrom  rather 
easily  by  using  the  proper  precautions,  as  described  in  the  original  puljli- 
cation. 

Zuelzer's  method  is  based  upon  the  precipitability  of  the  lecithin  by 
acetone,  and  Bergell's  ^  method  upon  the  preparation  of  the  double 
salt  of  cadmium  and  its  decomposition  by  ammonium  carbonate.  The 
preparations  oljtained  by  the  different  methods  consist  generally  of  a 
mixture  of  lecithins. 

The  detection  and  the  quantitative  estimation  of  lecithins  in  animal 
fluids  or  tissues  is  based  on  the  solubility  of  the  lecithins  (at  50-60°  C.)  in 
alcohol-ether,  by  which  the  phosphoric-acid  or  glycerophosphoric-acid 
salts  which  may  be  present  at  the -same  time  are  not  dissolved.  The 
alcohol-ether  extract  is  evaporated,  the  residue  dried  and  fused  with  soda 
and  saltpetre.  Phosphoric  acid  is  formed  from  the  lecithin,  and  it  can  be 
used  in  the  detection  and  quantitative  estimation.  The  distearyl-lecithin 
yields  8.798  per  cent  P2O5.  This  method  is,  however,  not  exactly  correct, 
for  it  is  possible  that  other  phosphorized  organic  combinations,  such  as 
jecorin  (see  Chapter  VIII)  and  protagon  (Chapter  XII),  may  have  passed 
into  the  alcohol-ether  extract.  In  detecting  lecithin  the  double  compovmd 
of  choline  and  platinum  chloride  must  also  be  prepared.  The  residue  of  the 
evaporated  alcohol-ether  extract  may  be  boiled  for  an  hour  with  baryta- 
water,  filtered,  the  excess  of  barium  precipitated  with  CO2,  and  filtered 
while  hot.  The  filtrate  is  concentrated  to  a  syrupy  consistency,  extracted 
with  absolute  alcohol,  and  the  filtrate  precipitated  with  an  alcoholic  solu- 
tion of  platinum  chloride.  The  precipitate  after  filtration  may  be  dissolved 
in  water  and  allowed  to  crystallize  over  sulphuric  acid.     For  the  detection 

'Chem.  Centralbl.,  1901,  2,  30  and  193. 

^Altmann,  cited  from  Hoppe-Seyler-Thierf elder's  Handbuch,  7.  Auflage;  Gilson, 
ibid. 

■■'  Zuelzer,  Zeitschr.  f.  physiol.  CIiem.,27,  and  Bergell,  Ber.  d.  d.chem.  Gesellsch.,  33. 


148  THE   ANIMAL  CELL. 

and  estimation  of  lecithin  we  can  make  use  of  the  method  of  heating  with 
hydriodic  acid  as  suggested  bv  Koch.^  One  methyl  iodide  group  is  split 
ofif  at  240°  and  the  two  others  at  about  300°  C. 

Protagons,  which  are  found  in  the  leucocytes  and  pus-cells,  are  also  to 
be  considered  as  constituents  of  protoplasm.  These  phosphorized  Ijodies 
occur  principally  in  the  brain  and  nerves,  and  hence  will  be  described  in  a 
following  chapter  (XII). 

Glycogen,  first  discovered  by  Cl.  Bernard,  is  found  in  developing 
animal  cells  and  especially  in  developing  embryonic  tissues.  According 
to  Hoppe-Seyler  it  seems  to  be  a  never-failing  constituent  of  the  cells 
which  show  ama?boid  movement,  and  he  found  this  carbohydrate  in  the 
leucocytes,  but  not  m  the  developed  motionless  pus-corpuscles.  Salomon 
and  afterwards  others  have,  however,  found  glycogen  in  pus.^  From  the 
relationship  which  seems  to  exist  between  glycogen  and  muscular  work  (see 
Chapter  XI),  it  is  presumable  that  a  consumption  of  glycogen  takes  place 
in  the  movement  of  animal  protoplasm.  On  the  other  hand,  the  extensive 
occurrence  of  glycogen  in  embryonic  tissues,  as  also  its  occurrence  in  patho- 
logical tumors  and  in  abmidant  cell  formation,  speaks  for  the  importance 
of  this  body  in  the  formation  and  development  of  the  cell. 

In  adult  animals  glycogen  occurs  as  stored  foodstuff  in  the  muscles  and 
certain  other  organs,  but  principally  m  the  liver;  therefore  it  will  be  com- 
pletely described  in  connection  with  this  organ  (Chapter  VIII). 

Another  body  or  perhaps  more  correctly  a  group  of  bodies  which  occur 
wideh'  distributed  in  the  animal  and  vegetable  kingdoms,  and  which  are 
present  regularly  in  the  cells,  are  the  cholesterins.  The  best-known  repre- 
.sentative  of  this  group  is  ordinan.-  choksterin  (see  Chapter  VIII),  which  is 
the  chief  constituent  of  certain  biliary  calculi  and  exists  in  abimdant  quan- 
tities in  the  brain  and  nerv-es.  It  is  hardly  probable  that  this  body  is  of 
direct  importance  for  the  life  and  development  of  the  cell.  It  must  be 
considered  that  the  cholesterin,  as  accepted  by  Hoppe-Seyler,^  is  a  cleavage 
product  appearing  in  the  cell  during  the  processes  of  life,  but  this  does  not 
exclude  the  possibility  that  the  cholesterin,  as  a  constituent  of  the  lipoids 
of  the  protoplasm-boundary  layers  (Overton),  may  be  of  indirect  im- 
portance in  cell  life.  According  to  Hoppe-Seyler.  the  same  is  true  for  the 
fats,  which  do  not  occur  constantly  in  the  cells  and  have  nothing  to  do  in 
the  ordinary  processes  of  life.  There  is  no  doubt  that  cholesterin  exists 
as  a  constituent  of  the  protoplasm,  but  its  existence  in  the  nucleus  is  ques- 
tionable. The  intracellular  enzymes  are  undoubtedly  constituents  of  the 
protoplasm  as  well  as  of  the  nucleus  and  must  be  of  the  greatest  import- 
ance for  the  life  and  fmictions  of  the  cells. 

'  Zeitschr.  f.  physiol.  Chem.,  36,  and  Amer.  Jour.  Physiol..  11. 
^  In  regard  to  the  literature  on  glycogen  see  Chapter  VIII. 
3  Physiol.  Chem.,  p.  81. 


XUCLEOPROTEIDS.  149 

The  cell  nucleus  has  a  rather  complex  structure.  It  consists  in  part 
of  fibrils  which  form  a  network  and  another  part  which  is  less  solid  and 
homogeneous.  The  first  differs  from  the  second  in  possessing  a  stronger 
affinity  for  many  dyes.  On  accoimt  of  this  behavior  the  first  is  called  the 
chromatic  substance  or  chromatin,  and  the  other  the  achromatic  substance 
or  achromatin. 

The  homogeneous  substance  of  the  nucleus  is  considered  as  a  mixture 
of  protein.  The  network  seems  to  contain  the  more  specific  constituent 
of  the  nucleus,  namely,  the  nuclein  substances.  Besides  this  it  is  alleged 
to  contain  another  substance  also,  plastin.  This  last  is  less  soluble  than 
the  nuclein  substances  and  does  not  have  the  property,  like  them,  of  fixing 
dyes. 

The  chief  constituents  of  the  cell  nucleus  are  the  nucleo'proteids,  and 
in  certain  cases  the  nucleic  acids. 

Nucleoproteids.  The  most  important  of  these  bodies  have  already 
been  discussed  in  a  previous  chapter  (II,  page  71).  These  bodies  are  either 
strong  or  loose  combinations  of  nucleic  acids  with  proteid.  To  the  latter 
class  belongs  histone,  in  certain  cases,  and  the  compounds  between  nucleic 
acids  and  protamines  should  also  perhaps  be  called  nucleoproteids.  There 
is  a  difference  among  the  nucleoproteids.  dependent  on  the  various  proteid 
complexes  as  well  as  upon  the  nucleic  acids.  They  contain  generally  con- 
siderable proteid  in  the  molecule,  hence  they  give  the  ordinary  proteid 
reactions,  and  therefore  are  closely  related  to  the  protein  bodies.  The 
nucleoproteids  occurring  in  the  cell  nucleus  seem  to  be  characterized  by 
containing  a  relatively  large  amount  of  phosphorus  and  a  pronoimced  acid 
character. 

In  the  preceding  discussion  of  the  nucleoproteids,  attention  was  called 
to  the  fact  that,  on  their  modification  b}'  heat,  b}'  weak  acid  action,  and 
by  peptic  digestion,  proteid  is  split  off  and  a  nucleoproteid  richer  in  phos- 
phorus is  formed.  These  compoimd  proteids,  rich  in  nucleic  acid,  obtained 
by  peptic  digestion  from  cells,  cell-rich  organs,  or  nucleoproteids.  have  been 
called  Jiuclcins  (Miescher,  Hoppe-Seyler  i)  or  true  nucleins.  But  as  the 
true  nuclein  seems  to  be  nothing  but  a  modified  nucleoproteid  poor  in 
proteid,  it  seems  unnecessaiy  to  give  the  name  nuclein  thereto.  On  the 
other  hand,  the  nucleins  have  other  properties  than  the  nucleoproteids, 
and  as  the  nucleins  bear  the  same  relationship  to  the  nucleoproteids  that 
the  pseudonuclein  does  to  the  nucleoalbumins,  we  will  give  here  a  short 
description  of  the  nucleins  as  well  as  the  pseudo-  or  paranucleins. 

Nucleins  or  true  nucleins  are  formed,  as  above  stated,  from  nucleo- 
proteids in  their  peptic  digestion  or  by  treatment  ^nth  dilute  acids.  It 
must   be  remarked  that   the  nucleins  are  not   entirely  resistant   towards 

'  Hoppe-Sej'ler ,  Med.  f^hem.  Untersuch.,  4.52. 


150  THE  ANIMAL  CELL. 

gastric  juice,  and  also  that  at  least  one  nucleoproteid,  namely,  the  one 
obtained  from  the  pancreas,  completely  dissolves,  lea\ing  no  nuclein  residue 
on  treatment  with  gastric  juice  (Umber,  Milroy').  The  nucleins  are 
rich  in  phosphorus,  containing  in  the  neighborhood  of  5  per  cent.  Accord- 
ing to  LiEBERMAXN,-  mctapliosphoric  acid  can  be  split  off  from  true  nucleins 
(yeast  nuclein).  The  nucleins  are  decomposed  into  proteid  and  nucleic 
acid  by  caustic  alkali,  and  as  different  nucleic  acids  exist,  so  also  there 
exist  different  nucleins.  As  previously  stated,  proteids  may  be  precipitated 
in  acid  solutions  by  nucleic  acids,  and  in  this  way,  as  shown  by  Milroy, 
combinations  of  nucleic  acid  and  proteids  may  be  prepared  which  behave 
quite  like  true  nucleins.  All  nucleins  yield  so-called  nuclein  bases  on  boil- 
ing with  dilute  acids.  The  nucleins  contain  iron  to  a  considerable  extent. 
They  act  like  rather  strong  acids. 

The  nucleins  are  colorless,  amorphous,  insoluble,  or  only  slightly  soluble 
in  water.  They  are  insoluble  in  alcohol  and  ether.  They  are  more  or  less 
readily  dissolved  by  dilute  alkalies.  The  nucleins  give  the  biuret  test  and 
MiLLOx's  reaction.  They  show  a  great  affinity  for  many  dyes,  especially 
the  basic  ones,  and  take  these  up  with  a\ddity  from  watery  or  alcoholic 
solutions.  On  burning  they  yield  an  acid  residue  which  is  ver}^  difficult 
to  incinerate  and  which  contains  metaphosphoric  acid.  On  fusion  with 
saltpetre  and  soda  the  nucleins  yield  alkali  phosphates. 

To  prepare  nucleins  from  cells  or  tissues,  first  remove  the  chief  mass  of 
proteids  by  artificial  digestion  with  pepsin-hydrochloric  acid,  lixiviate  the 
residue  with  very  dilute  ammonia,  filter,  and  precipitate  \^^th  hydrochloric 
acid.  The  precipitate  is  further  digested  with  gastric  juice,  washed  and 
purified  by  alternately  dissolving  in  very-  faintly  alkaline  water  and  re- 
precipitating  with  an  acid,  washing  ^^ith  water,  and  treating  ^^ith  alcohol- 
ether.  A  nuclein  may  be  prepared  more  simply  by  the  digestion  of  a 
nucleoproteid.  In  the  detection  of  nucleins  we  make  use  of  the  above- 
described  method,  testing  for  phosphorus  in  the  product  after  fusing  with 
saltpetre  and  soda.  Naturally  the  phosphates,  lecithins  (and  jecorin) 
must  first  be  removed  by  treatment  with  acid,  alcohol,  and  ether,  re- 
spectively. We  must  specially  call  attention  to  the  fact,  as  shown  by 
LiEBERMANN,^  that  it  is  very'  difficult  to  remove  lecithin  by  means  of 
alcohol-ether.  No  exact  methods  are  known  for  the  quantitative  estima- 
tion of  nucleins  in  organs  or  tissues. 

Pseudonucleins  or  Paraxucleixs.  These  bodies  are  obtained  as  an 
insoluble  residue  on  the  digestion  of  certain  nucleoalbumins  or  pliospho- 
glucoproteids  with  pepsin-hydrochloric  acid.  Attention  is  called  to  the 
fact  that  the  pseudonuclein  may  be  dissolved  by  the  presence  of  too  much 
acid  or  by  a  too  energetic  peptic  digestion.    If  the  relationship  between  the 

>  Umber,  Zeitschr.  f.  klin.  Med.,  43;    Milroy,  Zeitschr.  f.  physiol.  Chem.,  22. 
^  Pfliiger'.s  Arch.,  4". 
'  Ibid. 


NUCLEIC  ACIDS.  151 

degree  of  acidity  and  the  quantity  of  substance  is  not  properly  selected, 
the  formation  of  pseudonucleins  may  be  entirely  overlooked  in  the  digestion 
of  certain  nucleoalbumins.  Pseudonucleins  contain  phosphorus,  which, 
as  shown  by  Liebermaxx,^  is  split  off  as  metaphosphoric  acid  by  mineral 
acids. 

The  pseudonucleins  are  amorphous  bodies  insolul^le  in  water,  alcohol, 
and  ether,  but  readily  soluble  in  dilute  alkalies.  They  are  not  soluble  in 
\eTy  dilute  acids,  and  may  be  precipitated  from  their  solution  in  dilute 
alkalies  by  adding  acid.  They  give  the  protein  reactions  very  strongly, 
but  do  not  yield  nuclein  bases. 

In  preparing  a  pseudonuclein,  dissolve  the  mother-substance  in  hydro- 
chloric acid  of  1-2  p.  m.,  filter  if  necessary-,  add  pepsin  solution,  and  allow 
the  mixture  to  stand  at  the  temperature  of  the  body  for  about  twenty- 
four  hours.  The  precipitate  is  filtered  off,  washed  with  water,  and  purified 
by  alternately  dissohdng  in  very  faintly  alkaline  water  and  reprecipitating 
with  acid. 

Plastin.  After  the  extraction  of  the  nucleins  from  cell  nuclei  of  certain  plants 
by  dilute  soda  solution,  a  residue  is  obtained  which  is  characterized  by  its  great 
insolubility.  The  substance  which  forms  this  resiaue  has  been  called  plastin. 
This  substance,  of  which  the  spongioplasm  of  the  body  of  the  cell  and  the  nucleus 
granules  are  alleged  to  be  composed,  is  considered  as  a  nuclein  modification  of 
great  insolubility,  although  its  nature  is  not  known. 

Nucleic  Acids.  All  nucleic  acids  are  rich  in  phosphorus  and  yield  phos- 
phoric acid  and  nuclein  bases  as  cleavage  products.  The  various  nucleic 
acids  are  nevertheless  very  different  in  regard  to  the  products  they  yield. 
The  statements  in  this  regard  are  somewhat  contradictory  and  it  seems 
as  if  in  certain  cases  we  were  dealing  with  impure  or  partly  decomposed 
nucleic  acids.  For  example,  according  to  Kossel,  the  nucleic  acid  from 
ox-sperm  yields  chiefly  xanthine,  while  Levexe  obtained  only  guanine  and 
adenine.  The  guanylic  acid  isolated  by  Baxg  from  the  pancreas  contained 
only  guanine,  while  the  pancreas  nucleic  acid  investigated  by  Levexe 
contained  adenine  as  well  as  guanine.  The  nucleic  acids  of  the  thymus 
yield,  according  to  most  statements,  only  adenine  and  guanine,  similar  to 
the  acids  obtained  from  the  spleen,  brain,  mammar}'  gland,  and  fish-sperm. 
According  to  Steudel,  the  thymusnucleic  acids  yield  xanthine,  hypoxan- 
thine,  adenine,  and  guanine,while  according  to  Baxg  the  thymus  gland  con- 
tains two  different  nucleic  acids,  one  containing  adenine  and  guanine,  while 
the  other  contains  only  adenine,  hence  is  an  adenylic  acid.  The  nucleic  acid 
of  the  intestine  yields,  according  to  Ixouye  and  Kotake,  all  four  nuclein 
bases,  although  it  has  about  the  same  composition  as  the  salmonucleic 
acid,  W'hich  yields  only  adenine  and  guanine. 

All  nucleic  acids  thus  far  investigated,  with  the  exception  of  guanylic 

'  Ber.  d.  d.  chem.  Gesellsch.,  21,  and  Centralbl.  f.  d.  med.  Wissensch.,  1SS9. 


152  THE  ANIMAL  CELL. 

acid,  contain  also  representatives  of  the  pyrimidine  group;  there  seems  to 
exist  a  difference  in  this  regard  between  animal  and  plant  nucleic  acids. 
As  far  as  known,  in  the  plant  nucleic  acids  the  pyrimidine  group  is  repre- 
sented only  by  cytosine  and  uracil  (Kossel,  Ascoli,  Kossel  and  Steudel, 
Osborne  and  Harris),  and  in  the  ar.imal  (the  thymusnucleic  acids),  on 
the  contrary,  by  cytosine,  thymine,  and  uracil  (Kossel,  Neumann,  Levene). 
Mandel  and  Levene  ^  have  nevertheless  isolated  a  nucleic  acid  from 
haddock  eggs  which  yielded  uracil  but  no  thymine,  and  this  acid  behaved 
in  other  respects  like  a  nucleic  acid  from  plant-cells.  The  guanylic  acid 
contains,  as  above  remarked,  neither  uracil,  thymine,  nor  cytosine. 

The  nucleic  acids  show  a  different  composition  also  in  other  regards.  A 
reducing  pentose  group  can  be  split  off  from  guanylic  acid  and  the  vege- 
table nucleic  acids  (the  tritico-  and  yeast  nucleic  acid),  while  from  the  yeast 
nucleic  acid  also  a  hexose  is  claimed  to  be  obtained.  No  reducing  carbo- 
hydrate has,  on  the  contrary,  been  split  off  from  most  animal  nucleic  acids. 
Certain  observations  which  were  based  upon  qualitative  pentose  reactions 
seem  to  show  that  the  various  organs  contain  nucleoproteids  containing 
pentoses  and  that  we  have  several  nucleic  acids  which  yield  pentose  (see 
Chapter  III,  p.  110).  The  preparation  of  these  acids  in  a  pure  form  has 
been  attempted  only  in  a  few  cases,  and  the  qualitative  pentose  reactions 
are  not  to  be  relied  upon  to  any  great  extent.  Bang  ^  has  indeed  shown 
that  a  nucleic  acid  occurs  in  the  thymus  gland  which  gives  the  phloroglucin 
reaction  but  does  not  contain  any  pentose.  Those  nucleic  acids  which  do 
not  split  off  any  reducing  carbohydrate  contain  nevertheless  a  carbohydrate 
group  which,  as  Kossel  and  Neumann  first  showed,  on  deep  cleavage  with 
a  mineral  acid  3nelds  le\adinic  acid. 

We  generally  admit  of  4  atoms  of  phosphorus  in  the  empirical  formula? 
of  the  various  nucleic  acids.  In  salmonucleic  acid  the  relationship  of  phos- 
phorus to  nitrogen  is  as  4  to  14,  in  triticonucleic  acid  4  to  16,  and  in  guanylic 
acid  4  to  20.  The  form  of  combination  of  the  phosphorus  is  not  known 
with  positiveness,  but  it  seems  at  least  that  guanylic  and  triticonucleic 
acids  are  derivatives  of  a  pentahydroxylphosphorie  acid,  P(0H)5. 

1  Journ.  of  Biol.  Chem.,  1,  425,  and  Zeitschr.  f.  physiol.  Chem.,  49,  262. 

^  The  works  of  Kossel  and  his  pupils  on  nucleic  acids  are  found  in  Arch.  f.  (Anat.  u.) 
Physiol.,  1892,  1893,  and  1894;  Sitzungsber.  d.  Berl.  Akad.  d.  Wissensch.,  18,  1894,- 
Centralbl.  f.  d.  med.  Wissensch.,  1893;  Ber.  d.  deutsch.  chem.  Gesellsch.,  26  and  27; 
Zeitschr.  f.  physiol.  Chem.,  22  and  38.  See  also  Neumann,  Arch.  f.  (Anat.  u.)  Physiol., 
1S98  and  1899,  Suppl.;  Miescher,  Hoppe-Seyler's  Med.  chem.  Untersuch.,  441,  and 
Arch.  f.  exp.  Path.  u.  Pharm.,  3";  Schmiedeberg,  ibuL,  3"  and  43;  Osborne  and 
Harris,  Zeitschr.  f.  physiol.  Chem.,  36;  Bang,  ibid.,  26  and  31;  Hofmeister's  Beitrage,  o, 
and  Biochem.  Centralbl.,  1,  29.5;  Altmann,  Arch.  f.  (Anat.  u.)  Physiol.,  1899;  Ascoli, 
Zeitschr.  f.  physiol.  Chem.,  28  and  31;  Levene,  ibid.,  32,  3",  38,  39,  43,  and  45;  Man- 
del  and  Levene,  ibid.,  46,  47,  49,  iiO;  Inouye  and  Kotake,  ibid.,  46;  Steudel,  ibid.,  42, 
43,  46,  and  49. 


NUCLEIC  ACIDS.  153 

All  nucleic  acids  are  amorphous,  white,  and  have  an  acid  reaction. 
They  are  readily  soluble  in  aramoniacal  or  alkaline  water  and  form  insoluble 
salts  with  the  heavy  metals,  and  as  a  rule  also  insoluble  basic  salts  with  the 
alkaline  earths.  Guanylic  acid  is  soluble  with  difficulty  in  cold  water  but 
rather  readily  in  boiling  water,  from  which  it  separates  on  cooling.  Guanylic 
acid  is  readily  precipitated  from  its  alkali  compound  by  an  excess  of 
acetic  acid.  The  other  nucleic  acids  are,  on  the  contrary,  not  precipitated 
from  such  compomids  by  an  excess  of  acetic  acid,  but  by  a  slight  excess 
of  hydrochloric  acid,  especially  in  the  presence  of  alcohol.  In  acid  solu- 
tions these  latter  nucleic  acids  give  precipitates  with  proteids,  which  are 
considered  as  nucleins.  The  behavior  of  guanylic  acid  in  this  regard 
has  not  been  shown  on  account  of  the  great  difficulty  in  dissolving  this 
acid  in  dilute  acids.  All  nucleic  acids  are  insoluble  in  alcohol  and  ether. 
They  do  not  give  either  the  biuret  test  or  Millox's  reaction.  The  nu- 
cleic acids  are  optically  active  and  indeed  dextrorotatory  (Gamgee  and 
Jones  1). 

The  proteolytic  enzymes,  such  as  pepsin  and  trypsin,  decompose  the 
nucleoproteids  more  or  less;  the  nucleic  acids  are  not  split  by  these  enzymes 
as  far  as  phosphoric  acid  and  purine  bases.  Such  a  cleavage  can,  on  the 
contrary,  be  brought  about  by  erepsm  (Nakayama)  or  by  other  closely 
allied  enzymes  which  have  been  called  nucleases  (Iwaxoff,  Fr.  Sachs). 
Micro-organisms  can  also  bring  about  a  more  or  less  deep  cleavage  of  the 
nucleic  acids  (Schittexhelm  and  Schroter^). 

Guanylic  acid  differs  essentially  from  the  other  animal  nucleic  acids. 
These  latter  are  closely  related  to  each  other,  and  as  they  all  yield  thymine 
on  cleavage  and  in  this  regard  differ  markedly  from  the  guanylic  acid  and 
the  plant  nucleic  acids,^  they  can  for  the  present  be  treated  of  as  one  group 
which  has  received  the  common  name  of  thymonucleic  acids. 

Thymonucleic  Acids.  A.  Neumaxx  has  isolated  a-  and  ^-thymusnu- 
cleic  acids  from  the  thymus  gland.  The  a-acid  is  soluble  with  difficulty 
and  can,  according  to  Kostytschew,  be  transformed  (two  thirds),  with 
the  splitting  off  of  purine  bases,  into  the  .5-acid.  The  a-acid  gives  in 
proper  concentration  a  sodium  salt  which  gelatinizes  and  a  barium  salt 
which  is  precipitated  by  barium  acetate  in  substance  (Kostytschew). 
The  barium  salt  of  the  ,.5-acid  is  not  precipitated  by  barium  acetate.  Ac- 
cording to  Baxg.  the  thymus  contains  both  an  adenylic  acid  and  a  nucleic 
acid  which  contains  adenine  as  well  as  guanine.     This  last  acid  is  prob- 

'  Proceed.  Roy.  Soc,  72. 

-  Nakayama,  Zeitschr.  f.  physiol.  Chem...  41;  Iwanoff,  ibid.,  39;  Fr.  Sachs,  "1st  die 
Nuklease  mit  dem  Trj^jsin  identisch?"  Inaug.-Dissert.  Heidelberg,  1905;  Schitten- 
helm  and  Schroter,  Zeitschr.  f.  physiol.  Chem.,'  41. 

^See  Mandel  and  Levene,  Jour,  of  Biol.  Chem..  1.  425,  and  Zeitschr.  f.  physiol. 
Chem..  49. 


154  THE  ANIMAL  CELL. 

ably  the  thymusnucleic  acid  which  is  identical  with  the  nucleic  acid  from 
the  salmon  milt  (or  salmon iicleic  acid)  (Schmiedeberg  and  Herlant^). 

The  salmonucleic  acid  and  the  thymusnucleic  acid  as  obtained  by 
Schmiedeberg's  method  have  the  same  composition,  C40H56N14O16.2P2O6. 
Other  nucleic  acids,  such  as  those  prepared  by  Alsberg.  from  the  sperm  of 
the  burbot  (Lota  vulgaris),  and  by  Levene  from  ox  sperm,  brain,  and  spleen, 
are  identical  with  the  th3'musnucleic  acid  or  are  at  least  closely  related 
acids.  To  this  group  belong  also  the  nucleic  acids  from  the  kidneys  and 
the  mammary  glands  (Mandel  and  Levene),  from  the  intestinal  mucosa 
(Inouye  and  Kotake),  from  the  sperm  of  the  sturgeon  (Noll),  herring 
(Mathews,  Gulewitsch),  and  sea-urchin  (Mathews  2). 

On  the  decomposition  of  thymusnucleic  acids  (or  salmonucleic  acids)  in- 
termediate products  of  various  kinds  are  produced  by  a  more  or  less  com- 
plete cleavage  of  the  nuclein  bases.  One  of  these  is  thymic  acid,  which  is 
obtained  on  heating  the  free  acid  with  water  at  the  water-bath  tempera- 
ture, when  adenine  and  guanine  are  simultaneously  split  off.  Thymic  acid 
is  readily  soluble  in  water  and  yields  a  barium  salt  which  is  also  soluble  in 
water  and  has  the  formula  Ci6H23N3P20i2Ba  (Kossel  and  Neumann). 

On  cleavage  with  acids  first  a  part  of  the  nuclein  bases  is  split  off.  The 
remaining  part  is  more  difficult  to  set  free,  and  in  this  operation  an  abundant 
formation  of  melanin  and  a  decomposition  of  the  original  substance  take 
place  at  the  same  time.  When  one  half  of  the  purine  bases  have  been  split 
off  we  obtain  the  substance  called  heminucleic  acid  by  Alsberg,  which  con- 
tains onl}^  1  molecule  of  purine  bases  to  2  P2O5.  According  to  Schmiede- 
berg, tlwmusnucleic  acid  (or  salmonucleic  acid)  is  a  combination  of 
purine  bases  with  another  substance,  the  7iucleot in  phosphoric  acid, 
C30H46N4O15.2P2O5.  The  non-phosphorized  component  of  this  substance, 
the  nucJcoHn,  C30H42N4O13,  which  is  the  ground  substance  of  thymus- 
nucleic acid,  has  been  isolated  by  Alsberg.  On  the  decomposition  of 
nucleic  acids  with  5  per  cent  sulphuric  acid,  Levene  was  able  to  split  off 
the  purine  bases  completely  and  the  pyrimidine  bases  in  part.  The  car- 
bohydrate groups  went  completely  into  solution. 

KuTSCHER  and  Seemann  obtained  guanidine  and  urea,  but  no  uric  acid,  as 
products  on  the  oxidation  of  nucleic  acid  with  potassium  permanganate. 
KuTSCHER  and  Schenck  '  obtained  adenine,  oxalic  acid,  acetic  acid,  an  acid 
having  an  unknown  formula,  and  another  acid  which  they  call  martamic  acid, 
besides   guanidine    and    urea.      Martamic    acid   has   the    formula   CsHgNgOj  or 

^  Neumann,  1.  c;  Kostytschcw,  Zeitschr.  f.  I'hysiol.  Chem.,  39;  Bang,  Hofmeister's 
Beit  rage,  5;    Schmiedeberg  and  Herlant,  Arch.  f.  exp.  Path.  u.  Pharm.,  44. 

2  Alsberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  51;  Noll,  Zeitschr.  f.  physiol.  Chem.,  25; 
Mathews,  ibid.,  23;  Gulewitsch,  ibid.,  27;  see  also  for  the  other  references  foot-note  2, 
p.  152. 

^  Kutscher  and  Schenck,  Zeitschr.  f.  physiol.  Chem.,  44;  Kutscher  and  Seemann, 
Ber.  d.  d.  chem.  Gesellsch.,  36,  and  Centralbl.  f.  Physiol.,  17. 


NUCLEIC  ACIDS.  155 

CsHioXeO;,  and  gives  a  silver  salt  which  is  soluble  in  ammonia  or  nitric  acid,  and 
which  crystallizes  in  tufts  of  leaves.  The  crystalline  acid,  which  is  soluble  in 
ether,  sublimes  at  150°  and  does  not  give  the  murexide  test  or  Weidel's  test. 

Guanylic  Acid.  This  acid,  which  thus  far  has  been  obtained  only  from 
the  pancreas,  has,  according  to  B.a.xg,  the  composition  C44H66X20P4O34.  It 
is  readily  soluble  in  warm  water,  but  partialh-  separates  out  on  cooling.  It 
is  considered  as  an  ester  of  a  glycerophosphoric  acid  and  decomposes  on 
hydrolytic  cleavage  with  acids,  according  to  Bang,  into  4  molecules  of 
guanine,  3  molecules  of  pento.se  (/-xylose  according  to  Neuberg),  3  mole- 
cules of  glycerine,  and  4  molecules  of  phosphoric  acid. 

According  to  the  more  recent  investigations  of  Baxg  and  Raaschou  ^ 
the  guanylic  acid,  which  Baxg  now  designates  as  ,^-acid  is  formed,  in  the 
preparation  from  another  acid  called  a-guanylic  acid,  by  the  action  of  tlie 
alkali.  The  a-guanylic  acid,  which  is  readily  soluble  in  water,  even  in  cold 
water,  contains  less  phosphorus  and  nitrogen  (6.65  and  15.38  per  cent  re-' 
spectively)  as  compared  with  the  ,5-acid,  which  contains  7.64  per  cent  phos- 
phorus and  18.21  per  cent  nitrogen.  By  the  action  of  alkalies  the  a-guanylic 
acid  splits  off  a  pentose  group  and  is  converted  into  the  ,3-acid. 

The  following  acid  is  also  generally  included  among  the  nucleic  acids : 

Inosinic  acid,  C10H13N4PO8,  was  first  isolated  by  Liebig  from  the  flesh  of 
certain  animals  and  then  closely  studied  by  Haiser.^  It  contains  phosphorus, 
is  amorphous,  and  gives  crystalline  salts  with  barium  and  calcium.  Haiser 
obtained  hypoxanthine  as  a  cleavage  product  and  probably  also  trioxyvalerianic 
acid,  though  this  has  not  been  positively  proven. 

The  thvmusnucleic  acid  may  be  prepared  as  the  copper  salt,  according 
to  ScHMiEDEBERG.  from  the  heads  of  the  salmon  spermatozoa  or  from  the 
residue  after  the  peptic  digestion  of  the  thymus  glands  (Herlaxt).  The 
protamines  are  removed  by  the  action  of  copper  chloride  and  the  last  traces 
of  proteid  removed  by  dissolving  the  residue  in  dilute  caustic  potash  and 
precipitating  this  solution  with  alcohol,  and  this  is  repeated  until  it  fails  to 
give  the  biuret  test.  The  copper  salt  can  be  precipitated  by  copper  chloride 
from  the  water}'  solution  of  the  potassium  nucleate,  after  acidification  with 
acetic  acid.  According  to  Neumanx,  the  two  thymusnucleic  acids,  a  and  ,5, 
can  be  obtained  from  the  gland,  after  previously  boiling  the  same  with  water 
containing  acetic  acid  and  then  cutting  it  up  fine.  The  finely  divided  gland 
is  boiled  with  about  3  per  cent  NaOH  for  one-half  hour  for  the  a-acid  and 
two  hours  for  the  ^-acid.  and  sodium  acetate  is  added  at  the  same  time. 
After  neutralization  with  acetic  acid,  filtration  and  concentration,  the 
product  is  precipitated  with  alcohol.  The  nucleic  acids  can  be  obtained 
from  the  precipitated  sodium  nucleates  bj'  precipitating  with  alcohol  con- 
taining hydrochloric  acid.  In  the  separation  of  the  two  acids,  Kostytschew 
makes  use  of  tlie  difTerent  behavior  of  the  barium  salts  on  saturating  their 
sohition  Avith  barium  acetate  (see  aboveV     Levexe's^  method  consists,  on 

'  Hofmeister's  Beitrage,  4. 

^Liebig,  Annal.  d.  Chem.  u.  Pharm.,  62;    F.  Haiser,  Monatshefte  f.  Chem.,  16. 

^  Schmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  43;  Herlant,  ibid.,  44;    Neumann, 


156  THE  ANIMAL  CELL. 

the  contrary,  in  treating  the  organs  first  with  5  per  cent  sodium  hydrate  or 
Avith  S  per  cent  ammonia  in  the  cold,  then  nearly  neutralizing  -with  acetic 
acid,  precipitating  the  proteids  with  picric  acid,  and  treating  the  strongly 
acidified  liquid  (acetic  acid)  Avith  alcohol.  In  the  presence  of  sufficient 
acetate  the  nucleic  acids  are  precipitated.  ^lore  recently  Levene  has  sug- 
gested that  the  nucleic  acid  be  dissolved  in  strong  c^cetic  acid  and  then 
precipitated  A\'ith  copper  chloride  or  hydrochloric  acid. 

Guanylic  acid  may  be  best  prepared,  according  to  Bang  and  Raaschou, 
by  the  following  method:  After  treating  the  pancreas  with  1  per  cent 
sodium-hydrate  solution  for  twenty-four  hours  at  the  room  temperature, 
it  is  dissolved  by  warming,  then  made  faintly  acid  with  acetic  acid,  filtered, 
made  faintly  alkaline  with  ammonia,  strongly  concentrated,  and  precipi- 
tated \Adth  alcohol  while  hot.  The  proteoses  remain  in  solution,  and  the 
precipitated  guanylic  acid  (a-acid)  is  purified  by  repeated  solutions  in 
water  and  precipitations  by  alcohol. 

Plant  Nucleic  Acids.  Those  best  known  are  the  yeast  nucleic  acid  and  the  tri- 
ticonucleic  acid,  C41H61X16P4O3,,  isolated  by  Osborne  and  Harris  from  the  wheat 
embryo,  and  which  according  to  these  investigators  is  identical  with  the  yeast 
nucleic  acid.  The  plant  nucleic  acids  are  nearly  related  to  the  thymonucleic  acids, 
but  differ  from  them  by  the  fact  that  in  the  thymonucleic  acids  the  pyrimidine 
groups  are  represented  by  uracil,  cytosine,  and  thymine,  and  in  the  triticonucleic 
acid  by  cytosine  and  uracil.  This  last  acid,  which  is  dextrorotatory,  yields  on 
hydrolysis  with  acid  1  molecule  of  guanine,  1  molecule  each  of  adenine  and 
cytosine  (Wheeler  and  Johnson  ')>  2  molecules  of  uracil,  and  3  molecules  of  pen- 
tose for  every  4  atoms  of  phosphorus.  Levene  has  been  able  to  prepare  from 
the  tubercle  bacilli  nucleic  acids  whose  nature  has  not  been  closely  studied. 

Plasminic  acid  is  an  acid  which  was  prepared  [by  Ascoli  and  Kossel  ^  by 
the  action  of  alkali  upon  yeast.  It  contains  iron  and  is  soluble  in  very  dilute 
hydrochloric  acid  (1  p.  m.).  It  is  still  a  question  whether  it  is  a  mixture  or  a 
chemical  individual. 

In  regard  to  the  preparation  of  yeast  and  triticonucleic  acid  we  must  refer  to 
the  works  of  Altmann,  Kossel,  Osborne  and  Harris.^ 

Among  the  cleavage  products  of  the  nucleic  acids  the  purine  deriva- 
tives and  the  pyrimidine  derivatives  are  of  special  interest. 

Purine  Bases  (nuclein  bases,  alloxuric  bases,  xanthine  bodies).  With 
these  names  we  designate  a  group  of  bodies  consisting  of  carbon,  hydrogen, 
nitrogen,  and  in  most  cases  also  of  oxygen,  which,  by  their  composition, 
show  a  relationship  not  only  among  themselves,  but  also  with  uric  acid. 
All  these  bodies,  uric  acid  included,  are  considered  as  consisting  of  an 
alloxuric  and  a  urea  nucleus,  and  for  this  reason  Kossel  and  KRiJGER  have 
called  them  alloxuric  bases,  or  the  entire  group,  including  uric  acid,  alloxuric 
bodies.  According  to  E.  Fischer,^  who  has  not  only  shown,  in  several 
ways,  the  close  relationship  of  uric  acid  to  this  group,  but  has  also  pre- 

Arch.  f.  (Anat.  u.)  Physiol.,  1S99,  Supplb.;  Levene,  Zeitschr.  f.  physiol.  Chem.,  32 
and  4o;   Kostytschew,  ibid.,  39, 

■  Amer.  Chem.  Journ.,  25). 

2  Ascoli,  Zeitschr.  f.  physiol.  Chem.,  28 

^  See  foot-note  2,  p.  152. 

*iiee  Fischer,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30  and  32. 


PURINE  BASES.  157 

pared  a  number  of  the  members  of  this  group  synthetically,  they  are  all 

N=CH 

I  I 

derived  from  a  compound,  €5114X4  =  HC     C — NH  ,  called  'purine. 

II  II  >CH 
N— C— X 

The  different  purine  bodies  are  derived  therefrom  by  the  substitution  of  the 
various  hydrogen  atoms  by  hydroxyl,  amide,  or  alkyl  groups.  In  order  to  signify 
the  different  positions  of  substitution  Fischer  has  proposed  to  number  the  nine 
members  of  the  purine  nucleus  in  the  following  way: 

IX— C6 

2C   oC— X7 

1       I       >  C8. 
3X— C— X9 
4 
HX— CO 

For  example,  uric  acid,   OC     C — XH  ,  is  2,  6,  8-trioxypurine,  adenine, 

I       II  >C0 

HX— C— XH 
X=C.XH2  HX— CO 

II  II 

HC     C — XH  ,  is  6-aminopurine,  and  heteroxanthine,  OC     C — X.CH3   ,  is 

II      II  >CH  I        li       >CH 

X— C— X  HX— C— X 

7-methyl-2,  6-dioxjTDurine,  etc. 

The  starting-point  used  by  Fischer  for  the  synthetical  preparation  of  the 
purine  bases  was  2,  6,  S-trichlorpurine,  which  is  obtained,  with  8-oxy-2,  6-dichlor- 
purine  as  an  intermediary  product,  from  potassium  urate  and  phosphorus  oxy chlo- 
ride. The  close  relation  between  uric  acid  and  the  nuclein  bases  follows  from 
the  fact,  as  showm  by  Suxdvik,'  that  two  bodies  may  be  obtained  on  the  reduction 
of  uric  acid  in  alkaline  solution,  which,  although  not  quite  identical  with  xanthine 
and  hypoxanthine,  are  at  least  very  similar  thereto.  Gal'tier  claims  to  have 
prepared  xanthine  sjmthetically  by  heating  hydrocyanic  acid  with  water  and 
acetic  acid.     Further  syntheses  of  purine  bases  have  been  made  by  Traube.^ 

The  purine  bodies  or  alloxuric  bodies  found  in  the  animal  body  or  its 
excreta  are  as  follows:  Uric  acid,  xanthine,  heteroxanthine,  \-methylxanthine, 
paraxanthine,  guanine,  epiguanine,  hypoxanthine,  episarkine,  adenine,  and 
carnine.  The  bodies  theobromine,  theophylline,  and  caffeine,  occurring  in  the 
vegetable  kingdom,  stand  in  close  relationship  to  this  group. 

The  composition  of  the  purine  bodies  most  important  from  a  physio- 
logical standpoint  is  as  follows: 

Uric  acid,  C3H4X4O3 2,  6,  8-trioxypurine 

Xanthine,  C5H4N4O2 2,  6-dioxypurme 

1-methylxanthine,  C6H6N4O0 1-methyl  " 

Heteroxanthine,  C6H6N4O2 7-      " 

TheophyUine,  C7HgN402 1 ,  3-dimethyl         ' ' 

Paraxanthine,  C7HSN4O0 1,7- 

Theobromine,  C,HsN402 3,7- 

Caffeine,  C8H,oN402 1,  3,  7-trimethyl 

'  Zeitschr.  f.  physiol.  Chem.,  23. 

^  Gautier,  Compt.  rend.,  98,  1523,  and  Ber.  d.  deutsch.  chem.  Gesellsch.,  31;  W. 
Traube,  ibid.,  33,  and  Annal.  d.  Chem.  u.  Pharm.,  331. 


158  THE   ANIMAL  CELL. 

Hypoxanthine,  CgH^N^O 6-oxypurine 

Guanine,  C5H5N5O 2-amino        "      " 

Epiguanine,  CgH.Np 7-methyl   "      "  "      " 

Adenine,  C5H5N5.  . 6-aminop urine 

Episarkine,  C4HsN403(?) 

Carnine,  C7HSN4O3 

After  Salomon  ^  had  shown  the  occurrence  of  xanthine  bodies  in  young 
cells,  the  importance  of  the  xanthine  bodies  as  decomposition  products  of 
cell  nuclei  and  of  nucleins  was  sho'^Ti  by  the  pioneering  researches  of 
KossEL,  who  discovered  adenine  and  theophylline.  Kossel  gave  them 
the  name  nuclein  bases.  In  those  tissues  in  which,  as  in  the  glands,  the 
cells  have  kept  their  original  state,  the  nuclein  bases  are  not  found  free, 
but  in  combination  with  other  atomic  groups  (nucleins).  In  such  tissues, 
on  the  contrary,  as  in  muscles,  which  are  poor  in  cell  nuclei,  the  nuclein 
bases  are  found  in  the  free  state.  Since  the  nuclein  bases,  as  suggested  by 
Kossel,  stand  in  close  relationship  to  the  cell  nucleus,  it  is  easy  to  under- 
.stand  why  the  quantity  of  these  bodies  is  so  greatly  increased  when  large 
quantities  of  nucleated  cells  appear  in  such  places  as  were  before  relatively 
poorly  endowed.  As  an  example  of  this,  the  blood,  in  leucaemia,  is  ex- 
tremely rich  in  leucocytes.  In  such  blood  Kossel  ^  found  1.04  p.  m.  nuclein 
l:)ases,  against  only  traces  in  the  normal  blood.  That  the  nuclein  bases 
are  also  intermediate  steps  in  the  formation  of  urea  or  uric  acid  in  the 
animal  organism  is  probable,  and  vdW  be  showTi  later  (see  Chapter  XV). 

Only  a  few  of  the  nuclein  bases  have  been  found  in  the  urine  or  in  the 
muscles.  Only  four  bases — xanthine,  guanine,  hypoxanthine,  and  adenine 
— have  been  obtained,  thus  far,  as  cleavage  products  of  nucleins.  In 
regard  to  the  purine  bodies  from  other  substances  we  refer  the  reader  to 
their  respective  chapters.  Only  the  above  four  bodies,  the  real  nuclein 
bases,  will  be  considered  at  this  time. 

Of  these  four  bodies  xanthine  and  guanine  form  one  special  group  and 
hypoxanthine  and  adenine  another.  B3'  the  action  of  nitrous  acid  guanine 
is  converted  into  xanthine  and  adenine  into  hypoxanthine. 

C5H4N40.NH+HN02  =  C5H4N402  +  N2+H20; 

Guanine  Xanthine 

C5H4N4.NH  +HNO2  =  C5H4N4O  -'   N2  +  H2O. 
Adenine  Hypoxanthine 

Similar  transformations  may  be  brought  about  by  putrefaction  as  well 
as  by  the  action  of  special  enzymes.  The  researches  of  Schittenhelm, 
Levene,  Jones,  Partridge,  Winternitz,  and  Burian  ^  have  shown  that 
in  various  orjrans  desamination  enzymes,  such   as  giianase  and  adcnase, 

'  Sitzungsber.  d.  Bot.  Verein  der  Provinz  Brandenburg,  1880. 

-  Zeitschr.  f.  physiol.  Chem.,   7. 

^  See  Chapter  XV  (uric  acid  formation). 


XANTHINE.  159 

occur,  which  conveH  guanine  and  adenine  into  xanthine  and  hypoxanthine 
respectively,  and  also  oxidases  which  oxidize  hypoxanthine  into  xanthine 
and  this  then  into  uric  acid. 

On  cleavage  with  hydrochloric  acid  all  four  of  the  bodies  are  converted  into 
ammonia,  glycocoll,  carbon  dioxide,  and  formic  acid.  On  oxidation  with  hydro- 
chloric acid  and  potassium  chlorate,  xanthine,  bi"omadenine,  and  bromhypo- 
xanthine  yield  alloxan  and  urea;  guanine  yields  guanidine,  parabanic  acid  (an 
oxidation  product  of  alloxan),  and  carbon  dioxide.  According  to  Burian  '  the 
nuclein  bases  give  beautiful  ued  products  with  diazo-compounds  as  long  as  the 
imide  hydrogen  in  the  7th  position  (see  structural  formula  above)  is  not  substi- 
tuted. As  the  nucleic  acids  do  not  react  with  the  diazo  compounds,  Burian 
concludes  that  probably  the  nucleic-acid  residue  is  combined  with  the  imide  hy- 
drogen at  position  7. 

The  nuclein  bases  form  crystalline  salts  with  mineral  acids,  which,  with 

the  exception  of  the  adenine  salts,  are  decomposed  by  water.     They  are 

easily  dissolved  by  alkalies,  while  with  ammonia  their  action  is  somewhat 

different.     They  are  all  precipitated  from  acid  solution  by  phosphotungstic 

acid;  they  also  separate  as  silver  compounds  on  the  addition  of  ammonia 

and  ammoniacal  silver-nitrate  solution.     These  precipitates  are  soluble  in 

boiling  nitric  acid  of  1.1  specific  gravit3^     All  xanthine  bodies  are  also 

precipitated  by  Fehling's  solution  (see  Chapter  XV)  in  the  presence  of  a 

reducing  substance  such  as  hydroxy lamine  (Drechsel  and  Balke).     Copper 

sulphate  and  sodium  bisulphite  may  also  be  used  to  advantage  in  their 

precipitation  (KRtJGER).^     This  behavior  of  the  xanthine  bases  serves  just 

as  well  as  the  behavior  with  the  silver  solution  for  their  precipitation  and 

preparation. 

HN— CO 

II' 
Xanthine,  C5H4N407=  OC    C — NHv  (2,  6-dioxypurine),  is  found  in 

I       II  >CH 

HN— C—  N  ^ 

the  muscles,  liver,  spleen,  pancreas,  kidneys,  testicles,  carp-sperm,  thymus, 
and  brain.  It  occurs  in  small  quantities  as  a  physiological  constituent 
of  urine,  and  it  occasionally  has  been  found  as  a  urinarj^  sediment,  or 
calculus.  It  was  first  observed  in  such  a  stone  by  ^Iarcet.  Xantliine  is 
found  in  larger  amounts  in  a  few  varieties  of  guano  (Jar-vds  guano). 

Xanthine  is  amorphous,  or  forms  granular  masses  of  crystals,  or  may 
also,  according  to  Horbaczewski,^  separate  as  masses  of  shining,  thin, 
large  rhombic  plates  with  1  mol  water  of  cr}'stallization.  It  is  veiy  slightly 
soluble  in  water,  in  14  151-14  600  parts  at  16°  C,  and  in  1300-1500  parts 
at  100°  C.  (Almen*).     It  is  insoluble  in  alcohol  or  ether,  but  is  readily 

*  Ber.  d.  d.  chem.  Gesellsch.,  3". 

^  Balke,   Zur   Kenntnis   der  Xanthinkorper,   Inaug.-Diss.   Leipzig,    1893;   Kriiger, 
Zeitschr.  f.  physiol.  Chem.,  IS. 
^  Zeitschr.  f.  physiol.  Chem.,  23. 
*Journ.  f.  prakt.  Chem.,  9G. 


160  THE  ANIMAL  CELL. 

dissolved  by  alkalies  and  with  difficulty  by  dilute  acids.  With  hydro- 
chloric acid  it  gives  a  crystalline,  difficultly  soluble  combination.  With 
very  little  caustic  soda  it  gives  a  readily  crystallizable  compound,  which 
is  easily  dissolved  by  an  excess  of  alkali.  Xanthine  dissolved  in  ammonia 
gives  with  silver  nitrate  an  insoluble,  gelatinous  precipitate  of  silver  xan- 
thine. This  precipitate  is  dissolved  by  hot  nitric  acid,  and  by  this  means 
an  easily  soluble  crystalline  double  compound  is  formed.  Xanthine  in 
aqueous  solution  is  precipitated  on  boiling  with  copper  acetate.  At 
ordinary  temperatures  xanthine  is  precipitated  by  mercuric  chloride  and 
by  ammoniacal  basic  lead  acetate.  It  is  not  precipitated  by  basic  lead 
acetate  alone. 

When  evaporated  to  dryness  in  a  porcelain  dish  with  nitric  acid,  xan- 
thine gives  a  yellow  residue,  which  turns,  on  the  addition  of  caustic  soda, 
first  red,  and,  after  heating,  purple-red.  If  we  place  some  chloride  of  lime 
with  some  caustic  soda  in  a  porcelain  dish  and  add  the  xanthine  to  this 
mixture,  at  first  a  dark-green  and  then  quickly  a  brownish  halo  forms 
around  the  xanthine  grains  and  finally  disappears  (Hoppe-Seyler).  If 
xanthine  is  warmed  in  a  small  vessel  on  the  water-bath  with  chlorine- 
water  and  a  trace  of  nitric  acid,  and  evaporated  to  dryness,  and  the  residue 
is  then  exposed  mider  a  bell-jar  to  the  vapors  of  ammonia,  a  red  or  purple- 
violet  color  is  produced  (Weidel's  reaction).  E.  Fischer  ^  has  modified 
Weidel's  reaction  in  the  following  way:  He  boils  the  xanthine  in  a  test- 
tube  with  chlorine-water  or  with  hydrochloric  acid  and  a  little  potassium 
chlorate,  then  evaporates  the  liquid  carefully  and  moistens  the  dry  residue 
with  ammonia. 

HN— CO 

Guanine.  05115X50  =  H9N.C     C — NH        (2-amino-6-oxvpurine).     Gua- 

II     I!        >CH 

N— C— N^ 

nine  is  found  in  organs  rich  in  cells,  such  as  the  liver,  spleen,  pancreas, 
testicles,  and  in  salmon-sperm.  It  is  further  found  in  the  muscles  (in  very 
small  amounts),  in  the  scales  and  in  the  air-])ladder  of  certain  fishes  as 
iridescent  crystals  of  guanine-lime;  in  the  retinal  epithelium  of  fishes,  in 
guano,  and  in  the  excrement  of  spiders  it  is  found  as  chief  constituent.  It 
also  occurs  in  human  and  pig  urine.  Under  pathological  conditions  it  has 
been  found  in  leucsemic  blood,  and  in  the  muscles,  ligaments,  and  articula- 
tions of  pigs  with  guanine-gout. 

Guanine  is  a  colorless,  ordinarily  amorphous  powder  which  may  he 
obtained  as  small  crystals  by  allowing  its  solution  in  concentrated  am- 
monia to  spontaneously  evaporate.  According  to  Horbaczewski  it  may 
under   certain    conditions   appear    in   crystals   similar   to   creatinine   zinc 

'  Ber.  d.  deutsch.  chcni.  Gesellsch.,  30,  2236. 


GUANINE.     HYPOXANTHINE.  161 

chloride.  It  is  insoluble  in  water,  alcohol,  and  ether.  It  is  rather  easily 
dissolved  by  mineral  acids  and  readily  by  alkalies,  but  it  dissolves  with 
great  difficulty  in  ammonia.  According  to  Wulff  ^  100  c.c.  of  cold  am- 
monia solution  containing  1,  3,  or  5  per  cent  NH3  dissolve  9,  15,  or  10 
milligrams  of  guanine  respectively.  The  solubility  is  relatively  increased 
in  hot  ammonia  solution.  The  hydrochloride  readily  crystallizes,  and  has 
been  recommended  by  Kossel^  for  the  microscopical  detection  of  gua- 
nine, on  account  of  its  behavior  to  polarized  light.  The  sulphate  contains 
2  molecules  of  water  of  crystallization,  which  is  completely  expelled  on 
heating  to  120°  C,  and  this  fact,  as  well  as  the  fact  that  guanine  yields 
guanidine  on  decomposition  with  chlorine-water,  differentiates  it  from 
6-amino-2-oxypurin€,  which  is  considered  as  an  oxidation  product  of  adenine 
and  possibly  occurs  as  a  chemical  metabolic  product  (E.  Fischer).  The 
6-amino-2-oxypurine  sulphate  contains  only  1  molecule  of  water  of  crystalli- 
zation, which  is  not  expelled  at  120°  C.  Very  dilute  guanine  solutions  are  pre- 
cipitated by  both  picric  acid  and  metaphosphoric  acid.  These  precipitates 
may  be  used  in  the  quantitative  estimation  of  guanine.  The  silver  com- 
pound dissolves  with  difficulty  in  boiling  nitric  acid,  and  on  cooling  the 
double  compound  crystallizes  out  readily.  Guanine  acts  like  xanthine  in 
the  nitric-acid  test,  but  gives  with  alkalies  on  heating  a  more  bluish-violet 
color.  A  warm  solution  of  guanine  hydrochloride  gives  with  a  cold  satu- 
rated solution  of  picric  acid  a  yellow  precipitate  consisting  of  silky  needles 
(Capranica).  With  a  concentrated  solution  of  potassium  bichromate  a 
guanine  solution  gives  a  crystalline,  orange-red  precipitate,  and  with  a 
concentrated  solution  of  potassium  ferricyanide  a  yellowish-brown,  crystal- 
line precipitate  (Capranica).  The  composition  of  these  and  other  guanine 
compounds  has  been  studied  by  Kossel  and  Wulff.^  Guanine  does  not 
give  Weidel's  reaction. 

HN— CO 

I  I 

Hypoxanthine,  Sarkine,  C5H4N40  =  HC     C — NH         =(6-oxypurine). 

II  II        >CH 

N— C— N^ 

This  body  is  found  in  the  same  tissues  as  xanthine.  It  is  especially  abun- 
dant in  the  sperm  of  the  salmon  and  carp.  Hypoxanthine  occurs  also  in 
the  marrow  and  in  very  small  quantities  in  normal  urine,  and,  as  it  seems, 
also  in  milk.  It  is  found  in  rather  considerable  quantities  in  tlie  blood 
and  urine  in  leucaemia. 

Hypoxanthine  forms  very  small,  colorless,  crystalline  needles.     It  dis- 

'  Zeitschr.  f.  physiol.  Chem.,  17. 

^  Ueber  die  chem.  Zusammensetz.  der  Zelle,.Verh.  d.  physiol.  Gesellsch.  zu  Berlin, 
1890-91,  Nos.  5  and  6. 

^  Zeitschr.  f.  physiol.  Chem.,  17;    Capranica,  ibid.,  4. 


162  THE   ANIMAL   CELL. 

solves  with  difficulty  in  cold  water,  but  the  statements  in  regard  to  the 
solubility  therein  are  very  contradictory. ^  It  dissolves  more  readily  in 
boiling  water,  in  about  70-80  parts.  It  is  nearly  insoluble  in  alcohol,  but 
is  dissolved  by  acids  and  alkalies.  The  compound  with  h}-drochloric  acid 
is  crystalline,  and  is  more  soluble  than  the  corresponduig  xanthine 
derivative.  It  is  easily  soluble  in  dilute  alkalies  and  ammonia.  The  silver 
compound  dissolves  with  difficulty  in  boiling  nitric  acid.  On  cooling,  a 
mixture  of  two  hypoxanthine  silver-nitrate  compounds  possessing  an  in- 
constant composition  separates  out.  On  treating  this  mixture  with  am- 
monia and  an  excess  of  silver-nitrate  and  heating,  a  silver  hypoxanthine 
is  formed,  which  when  dried  at  120°  C.  has  a  constant  composition, 
2(C5H2Ag2N40)H20,  and  is  used  in  the  quantitative  estimation  of  hypo- 
xanthine. Hypoxanthine  picrate  is  soluble  with  difficulty,  but  if  a 
boiling-hot  solution  of  the  same  is  treated  with  a  neutral  or  only  faintly 
acid  solution  of  silver  nitrate  the  hypoxanthine  is  nearly  quantitatively 
precipitated  as  the  compound  C5H3AgX40.C6H2(N02)30H.  Hypoxan- 
thine does  not  yield  an  insoluble  compound  with  metaphosphoric  acid. 
WTien  treated,  like  xanthine,  with  nitric  acid  it  yields  a  nearly  colorless 
residue  which,  on  warming  with  alkali,  does  not  turn  red.  Hypoxanthine 
does  not  give  Weidel's  reaction.  After  the  action  of  hydrochloric  acid 
and  zinc  a  hypoxanthine  solution  becomes  first  ruby-red  and  then  brownish 
red  m  color  on  the  addition  of  an  excess  of  alkali  (Kossel).  According  to 
E.  Fischer  2  a  red  coloration  occurs  even  in  the  acid  solution. 
N=:C.NH2 

I     ! 

Adenine,  C5H5N5  =  HC    C — NHv  (6-aminopurine),  was  first  found 

1!     II  >H 

by  Kossel  3  in  the  pancreas.  It  occurs  in  all  nucleated  cells,  but  in 
greatest  quantities  in  the  sperm  of  the  carp  and  in  the  thymus.  Adenine 
has  also  been  found  in  leucsemic  urine  (Stadthagex  "*).  It  ma}'  be  obtained 
in  large  quantities  from  tea-leaves. 

Adenine  crystallizes  with  3  molecules  of  water  of  crystallization  in  long 
needles  which  become  opaque  gradually  in  the  air,  but  much  more  rapidly 
when  warmed.  If  the  crystals  are  warmed  slowly  with  a  quantity  of 
water  insufficient  for  solution,  they  become  suddenly  cloudy  at  53°  C,  a 
characteristic  reaction  for  adenine.  It  dissolves  in  1086  parts  cold  water, 
but  is  easily  soluble  in  warm.  It  is  insolul)le  in  ether,  but  somewhat 
soluble  in  hot  alcohol  and  easily  so  in  acids  and  alkalies.  It  is  more 
easily  soluble  in  ammonia  solution  than  guanine,  but  less  soluble  than 

'See  E.  Fischer,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30. 
^  Kossel,  Zeitschr.  f.  physiol.  Chem.,  12,  252;   E.  Fischer,  1.  c. 
'See  Zeitschr.  f.  physiol.  Chem.,  10  and  12. 
*Vircho'.v's  Ann.,  100. 


ADEMXE.  163 

hypoxanthine.  The  silver  compound  of  adenine  is  difficultly  soluble  in 
warm  nitric  acid,  and  deposits  on  cooling  as  a  ciystalline  mixture  of 
adenine  silver-nitrates.  ^Yith  picric  acid  adenine  forms  a  compound, 
C5H5N5.C6H2(N02)30H,  which  is  very  insoluble  and  which  separates  more 
readily  than  the  hypoxanthine  picrate  and  which  can  be  used  in  the  quanti- 
tative estimation  of  adenine.  We  also  have  an  adenine  mercurj'-picrate. 
Metaphosphoric  acid  with  adenine  gives  a  precipitate  which  dissolves  in 
an  excess  of  the  acid  if  the  solution  is  not  too  dilute.  Adenine  hydro- 
chloride gives  with  gold  chloride  a  double  compound  which  consists 
in  part  of  leaf-shaped  aggregations  and  in  part  of  cubical  or  prismatic 
crystals,  often  with  rounded  corners.  This  compound  is  used  in  the  micro- 
scopic detection  of  adenine.  With  the  nitric-acid  test  and  with  Weidel's 
reaction  adenine  acts  in  the  same  way  as  hypoxanthine.  The  same  is  true 
for  its  behavior  with  hydrochloric  acid  and  zinc  with  subsequent  addition 
of  alkali. 

The  procedure  for  the  preparation  and  detection  of  the  four  above- 
described  xanthine  bodies  in  organs  and  tissues  is,  according  to  Kossel  and 
his  pupils,  as  follows:  The  finely  divided  organ  or  tissue  is  boiled  for  three 
or  four  hours  with  sulphuric  acid  of  about  5  p.  m.  The  filtered  liquid  is 
freed  from  proteid  by  basic  lead  acetate,  and  the  new  filtrate  is  treated  with 
sulphuretted  hydrogen  to  remove  the  lead,  again  filtered,  concentrated,  and, 
after  adding  an  excess  of  ammonia,  precipitated  with  ammoniacal  silver 
nitrate.  The  silver  compound  (with  the  addition  of  some  urea  to 
prevent  nitrification)  is  dissolved  in  not  too  large  a  quantity  of  boiling 
nitric  acid  of  sp.  gr.  1.1,  and  this  solution  filtered  boiling  hot.  On  cool- 
ing, the  silver  xanthine  remains  in  the  solution,  while  the  double  com- 
pounds of  guanine,  hypoxanthine,  and  adenine  crj'stallize  out.  The 
silver  xanthine  may  1)6  precipitated  from  the  filtrate  by  the  addition  of 
ammonia  and  the  xanthine  set  free  by  means  of  sulphuretted  hydrogen. 
The  three  above-mentioned  sih'er-nitrate  compounds  are  decomposed  in 
water  with  ammonium  sulphide  and  heat;  the  silver  sulphide  is  filtered  off, 
the  filtrate  concentrated,  saturated  with  ammonia,  and  digested  on  the 
water-bath.  The  guanine  remains  undissolved,  while  the  other  two  bases 
pass  into  solution.  A  part  of  the  guanine  is  still  retained  by  the  silver 
sulphide,  and  may  be  li1)erated  by  boiling  it  with  dilute  hydrochloric  acid 
and  then  saturating  the  filtrate  with  ammonia.  A^  hen  the  above  filtrate 
containing  the  adenine  and  hypoxanthine,  which  has  been,  if  necessary, 
freed  from  ammonia  by  evaporation,  is  allowed  to  cool,  the  adenine  sepa- 
rates, while  the.  hypoxanthine  remains  in  solution.  According  to  Balke  ^ 
we  can  advantageously  precipitate  the  xanthine  bases  with  a  copper  salt  and 
hydroxylamine  as  abo\e  mentioned  and  then  further  separate  the  Ijodies. 
In  cases  where  the  proteids  have  not  been  completely  separated  it  is  advan- 
tageous to  precipitate  the  bases  as  copper  compounds  with  copper  sulphate 
and  bisulphite.  IvRtiGER  and  Schittexhklm's^  method  for  the  separation 
and  quantitative  estimation  of  purine  bodies  in  faeces  can  be  followed  and 
the  bases  then  transformed  into  silver  compounds. 

The  method  of  Buriax  and  Hall  ^  is  serviceable  in  the  estimation  of 

'Zeitschr.  f.  physiol.  Chom.    45.  'Ibid.,  3S. 


164  THE  ANIMAL  CELL. 

the  total  quantity  of  purine  bodies  in  animal  organs;  the  quantitative  estima- 
tion of  the  various  bases  is  performed  in  the  main  according  to  the  method 
above  described.  The  xanthine  is  weighed  as  silver  xanthine.  The  three 
silver-nitrate  compoimds  are  converted  into  the  corresponding  silver 
derivatives  by  ammonia  and  the  addition  of  silver  nitrate,  and  then  ammo- 
nium sulphide  is  allowed  to  act  upon  the  carefully  washed  silver  compounds. 
The  guanine  is  weighed  as  such.  The  ammoniacal  filtrate  containing  the 
adenine  and  hypoxanthine,  which  must  not  be  mixed  with  the  hydrochloric- 
acid  extract  of  the  silver  sulphide,  is  neutralized  and  a  cold  concentrated 
solution  of  sodium  picrate  added  until  the  entire  liquid  has  a  pronouncedly 
yellow  color.  The  adenine  picrate  is  immediately  filtered  off,  washed  on 
the  filter-paper  with  water,  dried  at  above  100°  C,  and  weighed.  The  fil- 
trate containing  the  h^'poxanthine  is  gradually  treated  while  boiling  hot 
with  silver  nitrate,  and  after  cooling  more  silver  nitrate  is  added  to  see  if 
the  precipitation  is  complete.  The  silver-hypoxanthine  picrate  is  washed, 
dried  at  100°  C,  and  weighed.  In  regard  to  the  composition  of  these  com- 
pounds see  pages  162  and  163.  This  method  of  separating  adenine  and 
hypoxanthine  presupposes  the  absence  of  hydrochloric  acid  in  the  liquid. 

The  above  method  of  separation  with  ammonia  does  not  give  exact 
results  on  account  of  the  not  inconsiderable  solubility  of  guanine  in  warm 
ammonia.  According  to  Kossel  and  Wulff.^  the  guanine  may  therefore 
be  precipitated  fj'om  sufficiently  dilute  solutions  by  an  excess  of  metaphos- 
phoric  acid  and  the  nitrogen  determined  in  the  washed  precipitate  by 
Kjeld.\hl's  method.  The  adenine  and  hypoxanthine  may  be  precipitated 
from  the  filtrate  by  ammoniacal  silver  nitrate.  The  silver  compound  is 
decomposed  with  very  dilute  hydrochloric  acid  and  the  adenine  separated 
from  the  hypoxanthine  according  to  the  suggestion  of  Bruhxs.^  In  regard 
to  the  complications  in  the  detection  and  exact  estimation  of  purine  bodies 
in  extracts  of  organs,  we  refer  to  the  works  of  His  and  Hagen  and  of 
BuRiAX  and  Hall.^ 

NH— CO 

I         I 
Uracil,  C4H4N202=OC        CH  (2,6-dioxypyrimidine),  was  first  obtained 

I         II 
NH— CH 

by  AscoLi  and  Kossel  from  yeast  nucleic  acid  and  later  prepared  by  Kos- 
sel and  Steudel  from  thymusnucleic  acid  and  herring  testicles,  b}'  Levexe 
from  the  spleen  and  pancreas  nucleic  acids,  and  by  Levexe  and  Maxdel  from 
the  nucleic  acid  of  the  haddock  roe.  The  synthetical  preparation  was  first 
performed  by  E.  Fischer  and  Roeder.^ 

Uracil  cr}'stallizes  in  needles  which  cluster  in  rosettes.  On  careful  heat- 
ing it  sublimes  in  part  undecomposed,  but  develops  red  vapors  and  decom- 
poses in  part.     It  is  readily  soluljle  in  hot  water  but  less  so  in  cold  Abater, 

'  Zeitschr.  f.  physiol.  Chem.,  1". 
2  76m/.,  14,  5.59. 

^  His  and  Hagen,  ibid.,  30,  and  Burian  and  Hall,  ibid.,  38. 

*Ascoli,  ibid.,  31;  Kossel  and  Steudel,  ibid.,  3";  Levene,  ibid.,  38,  39;  Levene 
and  Mandel,  ibid.,  49;    E.  Fischer  and  Roeder,  Ber.  d.  d.  chem.  Gesellsch.,  34. 


THYMINE.    CYTOSINE..  165 

and  is  nearly  insoluble  in  alcohol  and  ether.  It  is  readily  soluble  in  am- 
monia. It  is  precipitated  by  silver-nitrate  solution  only  after  the  careful 
addition  of  ammonia  or  bar>'ta-water,  as  the  precipitate  is  readily  soluble  in 
an  excess  of  ammonia.  Uracil  responds  to  Weidel's  test  (p.  160).  In 
regard  to  the  preparation  of  uracil  see  Kossel  and  Stel'del.i 

XH— CO 

I  I 

Thymine,  CoHeXoO-^^OC        C.CH3  (5-methvluracil;.    This  bodv.  which 

"    '         I 

XH— CH 

is  identical  with  nuclensin  obtained  by  ScHiHEDEBERG  from  salmonucleic  acid,. 

is  obtained  from  the  thymusnucleic  acids  and  was  first  prepared  by  Kossel 

and  X'eu^laxx  from  thymusnucleic  acid,  and  then  by  other  investigators, 

especially  Levexe  and  Maxdel.  from  other  animal  nucleic  acids.     Fischer 

and  RoEDER  and  recently  Gerxgross  -  have  prepared  it  s}Tithetically. 

Thymine  crv'stallizes  in  small  leaves  grouped  in  stellar  or  dendriform 
clusters  or.  rarely,  in  short  needles  (Gulewitsch  ^).  On  heating  it  sublimes. 
It  is  difficultly  soluble  in  cold  water,  more  soluble  in  hot  water,  and  insolu- 
ble in  alcohol.  It  behaves  like  uracil  towards  ammonia  or  bar}-ta-water 
and  silver  nitrate.  Thymine  is  precipitated  by  phosphotungstic  acid,  which 
does  not  precipitate  uracil.  Bromine-water  is  decolorized  by  thymine,  pro- 
ducing bromthymine.  For  its  detection  we  make  use  of  the  sublimation, 
the  beha\ior  towards  silver  nitrate,  and  its  elementarx*  analysis. 

In  regard  to  the   methods  of   preparation  see  Kossel  and  X'eumaxx 

and  W.  JoxES.-* 

HX— C.XH, 

I         II 
Cytosine,  C4H5X'30=OC       CH       (6-aniino-2-oxvpvrimidine) ,  was  first 

II 
N=CH 

prepared  by  Kossel  and  X'EUiL\xx  from  thymusnucleic  acid,  and  then  by 

Kossel  and  Steudel,  also  by  Levexe  and  ^Iaxdel,  from  the  spleen  and 

many  other  animal  nucleic  acids,  by  Ixouye  and  Kotake  ^  from  the  nucleic 

acid  of  the  intestine,  and  finalh^  also  by  Wheeler  and  Johxsox  from  tritico- 

nucleic  acid.    "Wheeler  and  Johxsox  ^  have  also  prepared  it  s^Tithetically. 

The  free  base  is    difficultly  soluble    in  water  and  cr}-stalhzes  in  thin 

leaves  with  a  mother-of-pearl  luster.     The  double  compound  ^rith  pla:i::um 

»Zeitschr.   f.  physiol.  Chem.,  37,   24.5. 

^  Schmiedeberg,  1.  c;  Arch.  f.  exp.  Path.  u.  Pharm.,  3";  Kossel  and  Neumann, 
Ber.  d.  d.  chem.  Gesellsch. ,  26  and  2";  Mandel  and  Levene.  Zeitschr.  f.  physiol.  Chem., 
46.  47,  49.  oO;  E.  Fischer  and  Roeder,  ibid..  34;  Gemgross,  iW<f.,  3S. 

^  Zeitschr.  f.  physiol.  Chem.,  27. 

*  Kossel  and  Neimaann,  1.  c,  and  W.  Jones,  Zeitschr.  f.  physiol.  Chem.,  29,  461. 

*In  regard  to  the  cited  works  see  foot-note  2,  p.  152. 

'Amer.  Chem.  Journ.,  29:    see  also  foot-note  2,  p.   1"2. 


166  THE   ANIMAL  CELL. 

chloride,  the  crystalline  picrate,  the  nitrate,  and  the  two  sulphates  are  of 
importance  in  the  detection  of  cytosine.  This  base  is  precipitated  by  phos- 
photungstic  acid  and  by  silver  nitrate  in  the  presence  of  an  excess  of 
barium  hydroxide,  which  fact  is  of  importance  in  the  detection  of  cytosine 
(Kutscher).  Cytosine  gives,  like  uracil,  the  murexid  reaction  with  chlorine- 
water  and  ammonia.  In  regard  to  the  preparation  of  this  base,  see 
KossEL  and  Steudel  and  Kutscher.^ 

The  purine  bases  and  the  pyrimidine  bodies  are  closely  related  to  each 
other  not  only  from  a  chemical  but  also  from  a  physiological  point  of  view, 
and  for  this  reason  the  question  has  been  repeatedly  asked  whether  or  not 
the  pyrimidine  bodies  might  not  in  part  be  products  produced  from  the 
purine  bodies  by  the  action  of  acid.  All  researches  thus  far  carried  on  to 
elucidate  this  question  contradict  such  a  possibility. 

Mineral  Bodies.  The  mineral  substances  found  habitually  in  the  cells 
of  higher  plants  and  of  animals  are  potassium,  sodium,  calcium,  magnesium, 
iron,  phosphoric  acid,  chlorine,  and  perhaps  also  iodine  (Justus).  In  cer- 
tain cells  we  also  find  manganese,  silicic  acid,  arsenic,  barium,  and  lithium.^ 
We  are  chiefly  indebted  to  Liebig  for  showing  that  the  mineral  bodies  are  as 
important  for  the  normal  constitution  of  the  organs  and  tissues,  as  well  as 
for  the  normal  performance  of  the  processes  of  life,  as  the  organic  constituents 
of  the  body.  The  importance  of  the  mineral  constituents  is  evident  from 
the  fact  that  we  know  no  animal  tissue  and  no  animal  fluid  which  is  free 
from  mineral  bodies,  and  also  from  the  fact  that  certain  tissues  or  tissue 
elements  contain  chiefly  certain  mineral  bodies  and  not  others.  In  regard 
to  the  alkali  compounds  this  division  is,  in  general,  as  follows:  The  so- 
dium compounds  occur  chiefly  in  the  fluids,  while  the  potassium  compounds 
occur  especially  in  the  form-elements.  Corresponding  to  this,  the  cells  con- 
tain chiefly  potassium  as  phosphate,  while  they  are  less  rich  in  sodium  and 
chlorine  compounds.  Still  we  have  some  exceptions  to  this  rule,  and  it 
must  be  remarked  that  Beebe  ^  has  found  considerably  more  sodium  than 
potassium  in  malignant  tumors. 

The  importance  of  potassium  for  the  life  and  the  development  of  the  cell 
has  been  shown  by  several  observations.  A  very  instructive  and  interesting 
example  of  this  action  has  been  shown  by  Loeb  *  in  his  investigations  on 
the  pathogenesis  of  the  egg  of  the  sea-annelide  Chsetoptenis.    The  un- 

'  Kossel  and  Steudel,  Zeitschr.  f.  physiol.  Chem.,  37  and  38;   Kutscher,  ibid..  38. 

^Justus,  Virchow's  Arch.,  170  and  176.  In  regard  to  arsenic  see  the  works  of 
Gautier,  Compt.  rend.,  129,  180,  131,  139;  Bertrand,  ibid.,  134;  Segale,  Zeitschr.  f. 
physiol.  Chem,,  42;  Kunkel,  ibid.,  44.  In  regard  to  the  barium  see  Schulze  and  Thier- 
felder,  Sitzungsber.  d.  Gesellsch.  naturforsch.  Freunde,  1905,  No.  1,  and  in  regard  to 
lithium  see  Hermann,  Pfliiger's  Arch.,  109. 

*Amer.  Journ.  of  Physiol.,  11  and  12. 

*Ibid.,  3,  4,  and   Pfliiger's  Arch.,  87. 


MINERAL  BODIES  OF  THE  CELLS.  167 

fertilized  eggs  could,  in  sea-water  alone,  develop  only  to  the  eighth  or  six- 
teenth cell  stage;  after  a  short  stay  in  sea-water  to  which  KCl  was  added 
they  developed  to  the  trichophora  larva.  The  fact  that  the  KCl  could  not 
be  replaced  by  other  chlorides,  but  could  be  replaced  by  other  potassium 
salts  also  shows  that  we  are  here  dealing  with  a  specific  action  of  the 
potassium  ions. 

The  division  of  the  potassium  in  cells  and  various  tissues  seems,  accord- 
ing to  Macallum,^  to  be  peculiar  and  essentially  different.  According  to 
]\1acallum  the  potassium  is  absent  in  the  cell  nuclei  and  in  the  head  of 
spermatozoa  as  well  as  in  nerve-cells  and  their  axis-cylinders,  while  it  occurs, 
on  the  contrary,  in  the  medullary  sheath  and  especially  in  the  region  of  the 
nodes  of  Ranvier.  A  peculiar  division  of  the  potassium  also  occurs  in  the 
muscle  fibers  and  secreting  glandular  cells. 

The  importance  of  phosphoric  acid  is  not  clear;  it  is  possible  that 
this  acid  is  important  for  the  formation  of  the  lecithins  and  nucleins,  and 
thereby  indirectly  makes  possible  the  processes  of  growth  and  division, 
which  are  dependent  upon  the  cell  nucleus.  Loew  ^  has  shown,  by  means 
of  cultivation  experiments  on  algse  Spirogyra,  that  only  by  supplying 
phosphate  (in  his  experiments  potassium  phosphate)  was  the  nutrition 
of  the  cell  nucleus  made  possible,  and  thereby  the  growth  and  division  of 
the  cells.  The  cells  of  the  Spirogyra  can  be  kept  alive  and  indeed  produce 
starch  and  proteins  for  some  time  without  a  supply  of  phosphates,  but 
their  growth  and  propagation  suffer. 

As  both  phosphoric  acid  and  iron  are  obtained  from  the  nuclein  sub- 
stances it  is  likely  that  these  mineral  bodies  are,  at  least  relatively,  richest 
in  the  nucleus.  As  to  the  division  of  the  mineral  bodies  between  the 
protoplasm  and  the  nucleus  we  know  nothing  with  positiveness,  and  the 
same  is  true  as  to  the  form  of  combination  of  the  mineral  bodies  in  the  nu- 
cleus. On  incineration  we  obtain  not  only  a  mixture  of  the  mineral  bodies 
of  the  nucleus  and  protoplasm,  but,  as  is  true  for  all  animal  fluids  and 
tissues,  the  original  relationship  is  markedly  changed.  The  combinations 
between  the  colloidal  and  mineral  substances  are  destroyed,  carbon  dioxide 
discharged,  and  sulphuric  acid  and  phosphoric  acid  may  be  produced  from 
the  organic  boches.  The  ordinary  chemical  analysis  is  not  sufficient  for 
the  study  of  the  mineral  constituents  of  the  fluids  or  tissue,  their  forms 
of  combination  and  action;  hence  we  must  resort  to  physical-chemical 
methods. 

According  to  the  investigations  carried  on  by  these  methods  the  con- 
clusion has  been  reached,  irrespective  of  the  importance  of  the  mineral 
bodies  for  the  osmotic  tension  in  the  cells  and  tissues,  that  the  part  taken 
by  the  mineral  bodies  in  cell  life  is  essentially  dependent  upon  the  action 

'  Journ.  of  Physiol.,  32.  ^  Biolog.  Centralbl.,  11,  269. 


168  THE  ANIMAL  CELL. 

of  the  ions.  For  example,  the  permeability  of  the  blood-corpuscles  as 
well  as  other  cells  for  neutral  alkali  salts,  which  will  be  treated  in  the 
following  chapter,  shows  an  exchange  of  ions.  The  investigations  of  Mail- 
LARU  on  the  toxic  action  of  copper  salts  and  of  Paul  and  Kornig^  on 
that  of  mercur}^  salts,  acids,  and  alkalies  offer  other  examples.  From  these 
investigations  it  follows  that  the  toxicity  is  dependent  upon  the  dissociation 
and  that  it  is  not  dependent  upon  the  total  amount  of,  for  example,  copper 
or  mercury  salts  present  in  the  solution,  but  rather  upon  the  number  of 
copper  or  mercury  ions. 

Beautiful  and  instructive  examples  of  the  importance  of  the  ions  for 
cell  life  have  been  shown  by  Loeb  2  and  his  collaborators.  It  is  not  within 
the  scope  of  this  book  to  give  a  detailed  account  of  this  important  work, 
but  perhaps  it  will  be  sufficient  to  give  at  least  one  example.  The  develop- 
ment of  the  eggs  of  the  Fundulus  can  be  retarded  for  a  long  time  by  a  f 
normal  NaCl  solution.  On  the  addition  of  CaS04  this  retardation  is  prevented 
and  the  development  proceeds.  Other  calcium  salts  act  like  the  sulphate, 
but  alkali  sulphates  like  Na2S04  or  other  neutral  alkali  salts  do  not  have 
this  action,  hence  it  must  be  a  calcium  ion  action.  Small  quantities  of 
other  divalent  cations,  also  trivalent  ions,  act  in  a  similar  way  to  calcium, 
while  the  salts  of  monovalent  cations  do  not  have  this  action.  The  fact 
that  the  fresh  fertilized  Fundulus  eggs  develop  in  distilled  water  as  well  as  in 
sea-water  shows  that  we  are  not  dealing  simply  with  a  taking  up  of  the  salts 
necessary  for  development,  but  rather  with  an  antagonistic  salt  action.  They 
quickly  die  in  a  pure  NaCl  solution  (having  a  concentration  equal  to  that 
of  sea-water);  but  if  to  the  NaCl  solution  a  small  amount  of  zinc  sul- 
phate is  added,  the  eggs  are  in  condition  to  form  an  embryo.  The  common 
salt  can  also  retard  the  toxic  action  of  the  zinc  salt.  According  to  Loeb 
every  solution  which  contains  only  one  electrolyte  is  poisonous,  and  this 
toxicity  can  be  prevented  by  another  electrolyte,  and  in  certain  cases  by 
two  other  electrolytes.  We  are  still  undecided  how  the  salts  act  in  this 
regard;  Loeb^  believes  that  the  antagonistic  action  of  two  salts  may  pos- 
sibly be  brought  about  by  the  fact  that  the  diffusion  in  the  egg  is  slower 
when  the  two  are  simultaneously  in  the  solution  than  when  each  is  alone 
in  the  solution.  It  is  a  difficult  question  to  decide  how  the  valence  of  the 
ions  influences  the  power  of  certain  ions  to  act  as  poisons  or  as  anti-poisons. 

The  chief  mass  of  the  cells  consists  of  colloids,  and  as  the  normal  func- 
tions of  the  cells  are  connected  with  a  certain  physical  condition  of  the  proto- 

'Maillard,  Journ.  de  Physiol,  et  Path.,  1;  Paul  and  Kronig,  Zeitschr.  f.  physikal. 
Chem.,  12,  and  Zeitschr.  f.  Hygiene,  25. 

^  Loeb.  Amer.  Journ.  Physiol.,  3,  4,  and  6;  Pfliiger's  Arch.,  80,  87,  88,  and  93  (with 
Gies)  97,  101,  and  107,  and  University  of  California  Publications,  Physiol.,  1  and  2. 
See  also  W.  Oswald,  Pfliiger's  Arch.,  106. 

'"  Pfliiger's  Arch.,  107. 


COLLOIDAL  CELL  SUBSTANCES.  169 

plasm  it  is  natural  to  consider  the  action  of  the  ions  in  relationship  to  the 
change  in  the  state  of  the  colloids.  The  colloids  can  be  precipitated  by 
electroh'tes,  and  the  investigations  of  H.irdy  and  Pauli  ^  show  that  we 
are  here  probabl}'  also  dealing  with  an  ion  action.  Negatively  charged 
colloids  are,  according  to  Hardy,  precipitated  by  cations  and  positively 
charged  by  anions.  A  physiologically  balanced  salt  mixture  suitable  for 
the  normal  functions  may  also  be  produced  by  the  antagonism  of  the  ion 
action  in  a  complex  solution  containmg  several  salts  (Loeb  and  Gies). 
Changes  in  one  or  the  other  direction  must  correspondingly  also  bring 
about  changes  in  the  state  of  the  colloid  by  the  action  of  the  ions.  The 
action  of  ions  in  these  cases,  as  well  as  the  nature  of  colloids  and  the  reasons 
for  the  change  in  their  conditions,  is  a  ver\^  difficult  question,  and  its  solu- 
tion is  still  not  answered.2 

'  See  foot-note  1  and  2,  p.  168,  and  Mathews,  Amer.  Joum.  of  Physiol.,  10  and  12. 

^  Hardy,  Journ.  of  Physiol.,  24,  and  Zeitschr.  f.  physikal.  Chem.,  33.  See  in  regard 
to  colloids  Hober,  Physikal.  Chemie  der  Zelle  und  der  Gewebe,  Leipzig,  1906.  Ham- 
burger, Osmotischer  Druck  und  lonenlehre  in  den  mediz.  Wissenschaften,  Bd.  3, 1904: 
and  H.  Aron,  Biochem.  Centralbl,,  3,  505,  and  4,  557. 


CHAPTER  YI. 
THE    BLOOD. 

The  blood  is  to  be  considered  from  a  certain  standpoint  as  a  fluid  tissue, 
and  it  consists  of  a  transparent  liquid,  the  blood-plasma,  in  which  a  vast 
number  of  solid  particles,  the  red  and  ivhite  blood-corpuscles  (and  the  blood- 
plates),  are  suspended.  We  also  find  in  the  blood  granules  of  different  kinds, 
which  are  to  be  considered  as  transformation  products  of  the  form-ele- 
ments.^ 

Outside  of  the  organism  the  blood,  as  is  well  known,  coagulates  more  or 
less  quickly;  but  this  coagulation  is  accomplished  generally  in  a  few  minutes 
after  lea^ing  the  body.  All  varieties  of  blood  do  not  coagulate  with  the 
same  degree  of  rapidity.  Some  coagulate  more  quickly,  others  more  slowly. 
In  vertebrates  with  nucleated  blood-corpuscles  (birds,  reptiles,  batrachia, 
and  fishes)  Delezexxe  has  sho'^n  that  the  blood  coagulates  very  slowly  if 
it  is  collected  under  precautions  so  that  it  does  not  come  in  contact  with 
the  tissues.  On  contact  with  the  tissues  or  with  tissue  extracts  it  coagu- 
lates in  a  few  minutes.  The  blood  "\nth  non-nucleated  blood-corpuscles 
(mammals)  coagulates,  on  the  contrar}-,  ver}'  rapidly.  The  coagulation 
of  the  blood  in  these  cases  may  also  be  somewhat  retarded  by  preventing 
the  blood  from  coming  in  contact  with  the  tissues  (Spaxgaro,  Arthus^). 
Among  the  varieties  of  blood  of  mammals  thus  far  investigated  the  blood 
of  the  horse  coagulates  most  slowly.  The  coagulation  may  be  more  or  less 
retarded  by  quickly  cooling;  and  if  we  allow  equine  blood  to  flow  directly 
from  the  vein  into  a  glass  cylinder  which  is  not  too  vdde  and  which  has  been 
cooled,  and  let  it  stand  at  0°  C,  the  blood  may  be  kept  fluid  for  several 
days.  An  upper  amber-yellow  layer  of  plasma  gradually  separates  from  a 
lower  red  layer  composed  of  blood-corpuscles  ^nth  only  a  little  plasma. 
Between  these  is  observed  a  whitish-gray  layer  which  consists  of  white 
blood-corpuscles. 

The  plasma  thus  obtained  and  filtered  is  a  clear  amber-yellow  alkaline 

'See  Latschenberger,  Wioo.  Sitzungsber. ,  lOo. 

'  Delezenne,  Compl  rend.  Soc.  de  biol.,  49,  Spangaio,  Aich.  ital.  do  Biol.  32; 
Arthur,  Jouro.  de  Physiol    et  Pathol.,  4. 

170 


BLOOD-PLASMA.  171 

(towards  litmus)  liquid  which  remains  fluid  for  some  time  when  kept  at 
0°  C,  but  soon  coagulates  at  the  ordinary  temperature. 

The  coagulation  of  the  blood  may  be  prevented  in  other  ways.  After 
the  injection  of  peptone,  or,  more  correctly,  proteose  solutions  into  the 
blood  (in  the  living  dog),  the  blood  does  not  coagulate  on  leaving  the  veins 
(Fano,  Schmidt-Mulheim  1).  The  plasma  obtained  from  such  blood  by 
means  of  centrifugal  force  is  called  peptone-plasma.  According  to  Arthus 
and  HuBER  ^  the  caseoses  and  gelatoses  act  similarly  to  fibrin  proteose  in 
dogs.  Eel  serum  and  certain  lymph-forming  extracts  of  organs  (see  Chapter 
VII)  also  have  an  analogous  action.  The  coagulation  of  the  blood  of  w-arm- 
blooded  animals  is  prevented  by  the  injection  of  an  effusion  of  the  mouth 
of  the  officinal  leech  or  a  solution  of  the  active  substance  of  such  an  infusion, 
herudin  (Franz),  into  the  blood  current  (Haycraft^),  If  the  blood  is 
allowed  to  flow  directly,  while  stirring  it,  into  a  neutral  salt  solution — best 
a  saturated  magnesium-sulphate  solution  (1  vol.  salt  solution  and  3  vols, 
blood)— we  obtain  a  mixture  of  blood  and  salt  which  remains  uncoagulated 
for  several  days.  The  blood-corpuscles,  which,  because  of  their  adhesive- 
ness and  elasticity,  would  otherwise  pass  easily  through  the  pores  of  the 
filter-paper,  are  made  solid  and  stiff  by  the  salt,  so  that  they  may  be  easily 
filtered.  The  plasma  thus  obtained,  which  does  not  coagulate  spontaneously, 
is  called  salt-plasma. 

An  especially  good  method  of  preventing  coagulation  of  blood  consists 
in  drawing  the  blood  into  a  dilute  solution  of  potassium  oxalate,  so  that 
the  mixture  contains  0.1  per  cent  oxalate  (Arthus  and  Pages  4).  The 
soluble  calcium  salts  of  the  blood  are  precipitated  by  the  oxalate,  and  hence 
the  blood  loses  its  coagulability.  On  the  other  hand,  Horne  ^  found  that 
chlorides  of  calcium,  barium,  and  strontium,  when  present  in  large  amounts 
(2-3  per  cent),  may  prevent  coagulation  for  several  days.  According  to 
Arthus  ^  a  non-coagulable  blood-plasma  may  be  obtained  by  drawing  the 
blood  into  a  sodium-fluoride  solution  until  it  contains  0.3  per  cent  NaFI. 

On  coagulation  there  separates  in  the  previously  fluid  blood  an  insoluble 
or  a  very  difficultly  soluble  protein  substance,  fibrin.  When  this  separation 
takes  place  without  stirring,  the  blood  coagulates  in  a  solid  mass  which, 
when  carefully  severed  from  the  sides  of  the  vessel,  contracts,  and  a  clear, 
generally  yellow-colored  liquid,  the  blood-serum,  exudes.  The  solid  coagulum 
which  encloses  the  blood-corpuscles  is  called  the  blood-clot  (placenta  san- 

'  Fano,  Arch,  f  (Anat.  u  )  Physiol,  1881;    Schmidt-Mulheim,  ihid.,  ISSO. 
^  Arch,  de  Physiol.  (5),  8. 

^Haycraft,  Proc.  Physiol.  Soc  ,  1884,  13,  and  Arch,  f  exp.  Path.  u.  Pharm.,  18; 
Franz,  Arch.  f.  exp.   Path.   u.   Pharm.,  49. 

*  Archives  de  Physiol.  (5),  2,  and  Compt.  rend.,  112. 

'Jcurn.  of  Physiol.,  19. 

'  Journ.  de  Physiol,  et  Pharm.,  3  and  4. 


172  THE  BLOOD. 

guinis).  If  the  blood  is  beaten  during  coagulation,  the  fibrin  separates  in 
elastic  threads  or  fibrous  masses,  and  the  defibrinated  blood  which  separates 
is  sometimes  called  cruor}  and  consists  of  blood-corpuscles  and  blood- 
serum,  while  uncoagulated  blood  consists  of  blood-coipuscles  and  blood- 
plasma.  The  essential  chemical  difference  between  blood-serum  and  blood- 
plasma  is  that  the  blood-serum  does  not  contain  even  traces  of  the  mother- 
substance  of  fibrin,  the  fibrinogen,  which  exists  in  the  blood-plasma,  and 
the  serum  is  proportionally  richer  in  another  body,  the  fibrin  ferment  (see 
page  175). 

I.    BLOOD-PLASMA  AND  BLOOD-SERUM. 
The  Blood-plasma. 

In  the  coagulation  of  the  blood  a  chemical  transformation  takes  place  in 
the  plasma.  A  part  of  the  proteins  separates  as  insoluble  fibrin.  The 
albuminous  bodies  of  the  plasma  must  therefore  be  first  described.  They 
are,  as  far  as  we  know  at  present,  fibrinogen,  nucleoproteid,  serglobulins,  and 
sfrnihumins. 

Fibrinogen  occurs  in  blood-plasma,  chyle,  lymph,  certain  transudates 
and  exudates,  in  bone-marrow  (P.  Muller),  and  perhaps  also  in  other 
lymphoid  organs.  The  seats  of  formation  of  fibrinogen  are,  according  to 
Mathew^s,  the  leucocytes,  especially  of  the  intestine,  according  to  ]\1uller, 
the  bone-marrow  and  probably  other  lymphoid  organs  such  as  the 
spleen  and  lymph  glands,  and  according  to  Doyon  and  Nolf,  the  liver. 
The  statement  that  the  intestinal  wall  is  a  seat  of  formation  of  fibrinogen,  a 
view  that  had  already  been  held  by  Dastre,  is  substantiated  not  only  by 
the  direct  researches  of  Mathews,  but  also  by  the  older  and  confirmed 
statement  that  the  blood  from  the  mesentery  vein  is  richer  in  fibrinogen 
than  the  arterial  blood.  The  occurrence  of  fibrinogen  in  the  bone-marrow, 
as  sho^Ti  by  oMuller,  and  an  increase  of  fibrinogen  in  the  blood  as  well  as  in 
the  bone-marrow  of  animals  immunized  wdth  certain  bacteria,  especially 
pus-staphylococci,  indicates  the  formation  of  fibrinogen  in  this  tissue.  That 
the  liver  takes  part  in  the  formation  of  fibrinogen  is  made  probable  by  the 
fact  that  the  quantity  of  fibrinogen  in  the  blood  strongly  diminishes  after 
the  extirpation  of  the  liver  (Nolf),  and  that  fibrinogen  may  indeed  be 
entirely  absent  in  the  blood  in  phosphorus  poisoning  (Corin  and  Ansiaux, 
Jacoby,  Doyon,  Morel,  and  Kareff^). 

^  The  name  cruor  is  used  in  different  senses.  We  sometimes  mean  thereby  only 
the  blood  when  coagulated  in  a  red  solid  mass,  in  other  cases  the  blood-clot  after 
the  separation  of  the  serum,  and  again  the  sediment  consisting  of  red  blood-corpuscles 
which  is  obtained  from  defibrinated  blood  by  means  of  centrifugal  force  or  by  letting 
it  stand. 

^  P.  Mijller,  Hofmeister's  Beitrage,  6;  Mathews,  Amer.  Journ.  of  Physiol.,  3;  Nolf, 
Bull.  Acad.  Roy.  Belg.,  1905,  and  Arch,  intern,  de  Physiol.,  3,  1905;  Corin  and  Ansiaux, 


FIBRINOGEN.  173 

FilDrinogen  has  the  general  properties  of  the  globulins,  but  differs  from 
other  globulins  as  follows:  In  a  moist  condition  it  forms  white  flakes  which 
are  soluble  in  dilute  common  salt  solutions,  and  which  easily  conglom- 
erate into  tough,  elastic  masses  or  lumps.  The  solution  in  5-10  per  cent 
NaCI  coagulates  on  heating  at  52-55°  C,  and  the  faintly  alkaline  or 
nearly  neutral  weak  salt  solution  coagulates  at  56°  C,  or  at  exactly  the 
same  temperature  at  which  the  blood-plasma  coagulates.  Fibrinogen 
solutions  are  precipitated  by  an  equal  volume  of  a  saturated  common 
salt  solution,  and  are  completely  precipitated  by  adding  an  excess  of  NaCl 
in  substance  (thus  differing  from  serglobulin).  A  salt-free  solution  of 
fibrinogen  in  as  little  alkali  as  possible  gives  with  CaCl2  a  precipitate  which 
contains  calcium  and  soon  becomes  msoluble.  In  the  presence  of  NaCl 
or  by  the  addition  of  an  excess  of  CaCl2  the  precipitate  does  not  appear.^ 
A  neutral  solution  of  fibrinogen  is  precipitated  b}'  a  concentrated  solution 
of  sodium  fluoride  when  added  in  sufficient  quantity.  Fibrinogens  from 
different  kinds  of  blood  behave  somewhat  differently  in  this  regard.  Ac- 
cording to  HuiSKAMP  2  fibrinogen  from  horse-blood  hardh'  dissolves  in  NaCl 
of  3-5  per  cent  at  ordinar}'  temperatures,  while  it  does  dissolve  at  40-45°. 
It  also  dissolves  in  ammonia  of  0.05  per  cent,  and  on  the  addition  of  3-5 
per  cent  NaCl  this  solution  can  be  neutralized.  The  fibrinogen  prepared 
by  HuiSKAMP  in  this  way  retained  its  t3'pical  properties.  Fibrinogen 
differs  from  the  myosin  of  the  muscles,  which  coagulates  at  about  the 
same  temperature,  and  from  other  protein  bodies,  in  the  property  of  being 
converted  into  fibrin  under  certain  conditions.  Fibrinogen  has  a  strong 
decomposing  action  on  hydrogen  peroxide.  It  is  quickly  made  insoluble 
by  precipitation  with  water  or  with  dilute  acids.  Its  specific  rotation  is 
(a)D=  — 52.5°  according  to  Mitti^lbach.^ 

Fibrinogen  may  be  easily  separated  from  the  salt-plasma  or  oxalate- 
plasma  by  precipitation  with  an  equal  volume  of  a  saturated  NaCl  solution. 
For  further  purification  the  precipitate  is  pressed,  redissolved  in  an  S  per 
cent  salt  solution,  the  filtrate  precipitated  by  a  saturated  salt  solution  as 
above,  and  after  being  treated  in  this  way  three  times,  the  precipitate  at 
last  obtained  is  pressed  between  filter-paper  and  finely  divided  in  water. 
The  fibrinogen  dissolves  with  the  aid  of  the  small  amouiit  of  NaCl  con- 
tained in  itself,  and  the  solution  may  be  made  salt-free  by  dialysis  with 
very  faintly  alkaline  water.  The  fibrinogen  can  be  nearly  freed  from 
fibrin-globulin,  which  will  be  spoken  of  later,  by  precipitating  with  double 
the  volume   of  saturated  sodium-fluoride  solution,  redissolving  in  water 

Maly's  Jahresber.,  24;  Jacoby,  Zeitschr.  f.  physiol.  Chem.,  30;  Doyon,  Morel,  and  Ivareff, 
Compt,  rend.,  140;    Doyon,  Morel,  and  Peju,  Compt.  rend.  soc.  biolog.,  58. 

'See  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22;    Cramer,  ibid.,  23. 

^Huiskamp,  ibid.,  44  and  46.  In  regard  to  fibrinogen  the  reader  is  referred  to 
the  author's  investigations.  Pfliiger's  Archiv,  19  and  22,  and  Zeitschr.  f.  physiol. 
Chem.,  28. 

^Zeitschr.  f,  physiol.  Chem.,  19. 


174  THE  BLOOD. 

with  0.05  per  cent  ammonia,  and  then  neutraUzing  this  solution  treated 
with  NaCl,  and  repeating  this  several  times.  Fibrinogen  may  also,  accord- 
ing to  Reye/  be  prepared  by  fractionall}-  precipitating  the  plasma  with  a 
saturated  solution  of  ammonium  sulphate.  We  have  no  knowledge  as  to 
the  purity  of  the  fibrinogen  so  prepared.  From  transudates  we  ordi- 
narily obtain  a  fibrinogen  which  is  strongly  contaminated  with  lecithin 
and  which  can  hardly  be  purified  without  decomposing  it.  The  methods 
for  the  detection  and  quantitative  estimation  of  fibrinogen  in  a  liquid  were 
formerly  based  on  its  property  of  yielding  fibrin  on  the  addition  of  a  little 
blood,  of  serum,  or  of  fibrin  ferment.  Reye  has  suggested  the  fractional 
precipitation  with  ammonium  sulphate  as  a  quantitative  method.  The  value 
of  this  method  has  not  been  sufficiently  tested. 

Fibrinogen  stands  in  close  relationship  to  its  transformation  product, 
fibrin. 

Fibrin  is  the  name  of  that  protein  body  which  separates  on  the  so-called 
spontaneous  coagulation  of  blood,  lymph,  and  transudates  as  well  as  in  the 
coagulation  of  a  fibrinogen  solution  after  the  addition  of  serum  or  fibrin 
ferment  (see  below). 

If  the  blood  is  beaten  during  coagulation,  the  fibrin  separates  in  elastic, 
fibrous  masses.  The  fibrin  of  the  blood-clot  ma}'  be  beaten  to  small,  less 
elastic,  and  not  particularly  fibrous  lumps.  The  typical  fibrous  and 
elastic  white  fibrin,  after  washing,  stands,  in  regard' to  its  solubility,  close 
to  the  coagulated  proteins.  It  is  insoluble  in  water,  alcohol,  or  ether.  It 
expands  in  hydrochloric  acid  of  1  p.  m.,  as  also  in  caustic  potash  or  soda 
of  1  p.  m.,  to  a  gelatinous  mass,  which  dissolves  at  the  ordmar}-  tempera- 
ture only  after  several  days;  but  at  the  temperature  of  the  ])ody  it  dis- 
solves more  readily  although  still  slowly.  Fibrin  may  be  dissolved  by 
dilute  salt  solutions  after  a  long  time  at  the  ordinary-  temperature  or  much 
more  readily  at  40°  C,  and  this  solution  takes  place,  according  to  Arthur 
and  HuBER  and  also  Dastre,^  without  the  aid  of  micro-organisms.  This 
action  is  due  to  proteolytic  enzymes  carried  down  by  the  fibrin  or  enclosed 
within  the  leucocytes  (Rulot^).  According  to  Greex  and  Dastre-*  two 
globulins  are  formed  in  the  solution  of  fibrm  in  neutral  salt  solution,  and 
according  to  Rulot  also  proteoses  (and  peptones)  on  the  solution  of  fibrin 
containing  leucocytes.  Fibrin,  like  fibrinogen,  decomposes  hydrogen 
peroxide,  due  to  a  contamination  with  catalases,  but  this  property  is  de- 
stroyed by  heating  or  by  the  action  of  alcohol. 

What  has  been  said  of  the  solubility  of  fibrin  relates  only  to  the  typical 
fibrin  obtained  from  the  arterial  blood  of  mammals  or  man  by  ■^^■hipping 

1  W.  Reye,  Uber  Nachweis  und  Bestimmung  des  Fibrinogens,  Inaug.-Diss.  Strass- 
burg,  1898. 

^  Arthus  and  Huber,  Arch,  de  Physiol.   (5),  5;    Dastre,  ibid.  (5),  7. 

^  Arch,  intern,  de  Physiol.,  1. 

^  Green,  .Jour.i.  of  Fhysiol.,  8;    Dastre,  1.  c. 


FIBRIN.  175 

and  washing  first  with  water  and  with  common  salt  solution  and  then 
with  water  again.  The  blood  of  various  kinds  of  animals  yields  fibrm  with 
somewhat  different  properties,  and  according  to  FeR.\u  ^  pig-fibrin  dissolves 
much  more  readily  than  ox-fibrin  in  hydrochloric  acid  of  5  p.  m..  Fibrins 
of  varying  purity  or  originatmg  from  blood  from  different  parts  of  the  body 
have  unlike  solubilities. 

The  fibrin  obtained  by  beating  the  blood  and  purified  as  above  de- 
scribed is  always  contaminated  by  secluded  blood-corpuscles  or  remains 
thereof,  and  also  by  lymphoid  cells.  It  can  be  obtained  pure  only  from 
filtered  plasma  or  filtered  transudates.  For  the  pure  preparation,  as  well 
as  for  the  quantitative  estimation  of  fibrui,  the  spontaneously  coagu- 
lating liquid  is  at  once,  or  the  non-spontaneously  coagulatmg  liquid  orly 
after  the  addition  of  blood-serum  or  fibrin  ferment,  thoroughly  beaten 
with  a  whalebone,  and  the  separated  coagulum  is  washed  first  in  water 
and  then  with  a  5  per  cent  common  salt  solution,  and  again  with  water, 
and  finally  extracted  with  alcohol  and  ether.  If  the  fibrin  is  allowed  to 
stand  for  some  time  in  contact  with  the  blood  from  which  it  was  formed, 
it  partly  dissolves  ifibrinoh/sis — Dastre^).  This  fibrinolysis  must  be 
prevented  in  the  exact  quantitative  estimation  of  fibrin  (Dastre).  The 
blood  constituents  that  are  active  in  fibrinolysis  are  still  not  known,  but 
they  are  without  doubt  of  enzymotic  nature.  It  must  be  mentioned  that 
a  strong  fibrinolysis  takes  place  in  blood  after  acute  phosphorus-poisoning 
(Jacoby  and  others),  after  extirpation  of  the  liver  (Nolf),  and  also  when 
the  coagulability  of  the  blood  has  been  reduced  by  the  injection  of  pro- 
teoses (Nolf,  Rulot^). 

A  pure  fibrinogen  solution  may  be  kept  at  the  ordinary'  temperature 
until  putrefaction  begms  without  showmg  a  trace  of  fibrin  coagulation. 
But  if  to  this  solution  is  added  a  water-washed  fibrin-clot  or  a  little  blood- 
serum,  it  immediately  coagulates  and  may  yield  perfectly  typical  fibrin. 
The  transformation  of  the  fibrinogen  into  fibrm  requires  the  presence  of 
another  body  contained  in  the  blood-clot  and  in  the  serum.  This  body, 
whose  importance  in  the  coagulation  of  fibrin  was  first  observed  by 
BucHAXAX.^  was  later  rediscovered  by  Alexaxder  Schmidt^  and  desig- 
nated as  fibrin  ferment  or  thrombin.  The  nature  of  this  enzymotic  body  has 
not  been  ascertained  with  certainty.  Although  many  investigators, 
especially  English,  consider  fibrin  ferment  as  a  globulin,  still  more  recent 
experiments  of  Pekelh.\eixg  and  others  show  that  it  is  a  nucleoproteid 
which   according  to  Huiska.mp  ^   occurs   in   the   thymus  gland   partly   as 

'  Zeitschr.  f.  Biologic,  28. 

-  Archives  cle  Physiol.  (.5),  5  and  6. 

^Jacoby,  Zeitschr.  f.  physiol.  Chem.,  30;  Xolf,  Arch,  intern,  de  Physiol..  3,  1905; 
Rulot,  1.  c. 

^London  Med.  Gazette,  1S4.5,  617.     Cit.  by  Gamgee,  Journal  of  Physiol.,  1879. 

^Pfliiger's  Arch.,  G;  see  also  Zur  Blutlehre,  1892,  and  Weitere  Beitriige  zur  Blut- 
lehre,  1895. 

'  Pekelharing,  Verhandl.  d.  kon.  Akad.  d.  Wetensch.  te  Amsterdam,  1892,  Deel  1; 


176  THE  BLOOD. 

nucleohistone  and  partly  in  another  form.  Fibrin  ferment  is  produced, 
according  to  Pekelharing,  by  the  influence  of  soluble  calcium  salts  on  a 
preformed  zymogen  existing  in  the  non-coagulated  plasma.  ScH^nDT 
admits  of  the  presence  of  such  a  mother-substance  of  the  fibrin  ferment 
in  the  blood  and  calls  it  prothrombin.  The  conversion  of  this  mother- 
substance  into  thrombin  requires,  according  to  more  recent  investigations, 
the  presence  of  a  second,  zymoplastic-acting  substance  (see  Coagulation 
of  the  Blood).  Thrombin  is  like  other  enzymes  in  that  the  ver>'  smallest 
amount  of  it  produces  an  action  and  its  solution  becomes  inactive  on. 
heating.  The  velocity  of  coagulation  is  dependent  upon  the  quantity 
of  thrombin,  and  Fuld  has  found  that  at  least  within  certain  limits  an 
increase  of  double  the  quantity  of  enzyme  causes  an  increase  of  the  coagu- 
lation velocity  to  one  and  one  half.  This  is  true  only  for  experiments  with 
plasma  and  solutions  containing  kinases  (see  Coagulation  of  the  Blood),  and 
Martin  ^  has  found  another  law  from  experiments  with  plasma  and  snake- 
poisons  containing  thrombin.  According  to  him  the  behavior  is  as  follows: 
As  in  the  casein  coagulation  with  rennin,  the  velocity  of  coagulation  is 
inversely  proportional  to  the  quantity  of  ferment.  The  optimum  of  the 
thrombin  action  lies  at  about  40°  C;  at  70-75°  C.  the  enzyme  is  destroyed. 
The  question  as  to  whether  the  thromljin  found  in  different  animals  is 
the  same  substance  or  whether  we  have  several  thrombins  has  not  been 
decided.  The  latter  is  not  improbable;  nevertheless  a  definite  specificity 
of  different  thrombins  has  not  been  observed  with  certainty. 

The  isolation  of  thrombin  has  been  tried  in  several  ways.  Ordinarily 
it  may  be  prepared  by  the  following  method,  proposed  by  Alex.  Schmidt.^ 
Precipitate  the  serum  or  defibrinated  blood  with  15-20  vols,  of  alcohol  and 
allow  it  to  stand  a  few  months.  The  precipitate  is  then  filtered  and  dried 
over  sulphuric  acid.  The  ferment  may  be  extracted  from  the  dried 
powder  by  means  of  water.  Other  methods  have  been  suggested  by 
Hammarsten  and  by  Pekelharing.^ 

The  preparation  of  a  thrombin  solution  as  free  as  possible  from  lime 
may  be  accomplished  by  removing  the  lime  salts  from  the  serum  by  means 
of  oxalate  and  precipitating  the  serum  with  alcohol  and  allowing  it  to 
stand  under  alcohol  for  several  months.  The  dried  powder  is  rubbed  with 
water  and  freed  from  soluble  salts  by  repeated  lixiviation  with  water  and 
by  the  use  of  centrifugal  force.  Then  each  gram  of  powder  is  allowed  to 
stand  some  time  with  100-150  c.c.  water,  is  filtered,  and  in  this  way  a  solu- 

ibid.,  1895,  and  Centralbl.  f.  Physiol.,  9;  Wright,  Proc.  Roy.  Irish  Acad.  (.3),  2,  The 
Lancet,  1892,  and  On  Wooldridge's  Method,  etc.,  British  Med.  Journal,  1891;  Lilien- 
feld,  Hamatol.  Untersuch.,  Arch.  f.  (Anat.  u.)  physiol.,  1892;  Uber  Leukocyten  und 
Blutgerinnung,  ibid.;  Halliburton  and  Brodie,  Journal  of  Physiol.,  17  and  18;  Huiskamp, 
Zeitschr.  f.  physiol.  Chem.,  32;    Pekelharing  and  Huiskamp,  ibid.,  39. 

'Martin,  Journ.  of  Physiol.,  32;    Fuld,  Hofmeister's  Beitrage,  2. 

^  Pfliiger's  Arch.,  6. 

'  Hammarsten,  ibid. ,  18;   Pekelharing,  1.  c. 


COAGULATION   OF  FIBRINOGEN.  177 

tion  is  obtained  which  contains  only  about  0.3-0.4  p.  m.  solids  and  about 
0.0007  p.  m.  CaO  (Hamaluisten). 

If  a  fibrinogen  solution  containing  salt,  as  above  prepared,  is  treated 
with  a  solution  of  fibrin  ferment,  it  coagulates  at  the  ordinary  tempera- 
ture more  or  less  quickly  and  yields  a  typical  fibrin.  Besides  the  fibrin 
ferment  the  presence  of  neutral  salts  is  necessary,  for  Alex.  Schmidt  has 
shown  that  fil^rin  coagulation  does  not  take  place  without  them.  The 
presence  of  soluble  calcium  salts  is  not,  as  is  generally  assumed,  a  positive 
condition  for  the  formation  of  fibrin,  because,  as  shown  by  Alex.  Sch\udt, 
Pekelharixg,  and  Hammarstex,^  thrombin  can  transform  fibrinogen  into 
typical  fibrin  in  the  absence  of  lime  salts  precipitable  by  oxalate.  The 
fibrin  is  not  richer  in  lime  than  the  fibrinogen  (Hammarstex)  used  to  pre- 
pare it  if  the  fibrinogen  and  thrombin  solutions  are  employed  as  lime-free  as 
possible,  and  the  view  that  the  fibrin  formation  is  connected  with  a  taking 
up  of  lime  has  been  shown  to  be  untenable.  The  quantity  of  fibrin  obtained 
on  coagulation  is  always  smaller  than  the  amount  of  fibrinogen  from  which 
the  fibrin  is  derived,  and  we  always  find  a  small  amount  of  protein  substance 
in  the  solution.  It  is  therefore  not  improbable  that  the  fibrin  coagulation, 
in  accordance  with  the  views  first  proposed  by  Dexis,  is  a  cleavage  process 
in  which  the  soluble  fibrinogen  is  split  into  an  insoluble  protein,  the  fibrin, 
which  forms  the  chief  mass,  and  a  soluble  protein  substance  which  is  pro- 
duced only  in  small  amounts.  We  find  a  globulin-like  substance  which 
coagulates  at  about  64°  C.  in  blood-serum  as  well  as  in  the  serum  from 
coagulated  fibrinogen  solutions.  This  substance  is  called  fibrin-globulin  by 
Hammarstex.  The  recent  investigations  of  Huiskamp  have  sho^\ii  that  this 
substance  is  not  formed  as  a  cleavage  product  from  pure  fibrinogen  but  occurs 
in  plasma  or  in  fibrinogen  solutions  not  purified  of  sodium  fluoride  beside 
the  fibrinogen,  or  perhaps  in  loose  combination  with  fibrinogen.  The  view 
that  a  cleavage  takes  place  in  the  coagulation  of  the  fibrinogen  has  not 
been  supported  by  these  investigations.^ 

There  exist  also  other  views  in  regard  to  the  processes  of  coagulation  in 
the  formation  of  fibrin  which  are  even  less  positively  founded.  The  fact 
that  the  soluble  lime  salts  are  not  necessary  for  the  transformation  of  fibrin- 
ogen into  fibrin  is  not  in  contradiction  to  the  other  fact  that  they  must  be 
present  in  the  coagulation  of  blood  or  plasma.  This  apparent  contradiction 
may  be  explained,  as  shown  later,  by  the  special  condition  of  the  blood- 
plasma,  and  we  must  not  overlook  the  fact  that  the  coagulation  of  the  blood 
is  a  much  more  complicated  process  than  the  coagulation  of  a  fibrinogen 


'See  Hammarsten,  Zeitschr.  f.  physioI.Chem.,  22,  which  also  cites  the  works  of 
Schmidt  and  Pekelharing,  and  ibid.,  28. 

^  See  Hammarsten,  Zeitschr.  f.  phj'siol.  Chem.,  2S;  Heubner,  Arch,  f,  exp.  Path, 
u.  Pharm.,  49,  and  Zeitschr.  f.  physiol.  Chem.,  4.5;   Huiskamp,  ibid.,  44  and  46, 


178  THE  BLOOD. 

solution,  inasmuch  as  the  first  involves  other  important  questions,  as,  for 
instance,  the  reason  for  the  blood  remaming  fluid  in  the  body,  the  origin  of 
the  fibrin  ferment,  the  importance  of  the  form-elements  in  the  coag-ulation 
etc.     A  fuller  discussion  of  the  various  hypotheses  and  theories  concerning 
the  coagulation  of  the  blood  must  therefore  be  given  later. 

Nucleoproteid.  This  substance,  which,  as  above  mentioned,  is  considered 
by  Pekelharing  and  Huiskamp  as  identical  with  the  prothrombin  or  thrombin, 
occurs  in  the  blood-plasma  as  well  as  in  the  serum,  and  is  precipitated  from  the 
latter  with  the  globulin.  It  is  similar  to  the  globulin  in  that  it  is  readily  soluble 
in  neutral  salt  solution  and  can  be  completely  salted  out  on  saturation  with 
magnesium  sulphate  and  separates  only  incompletely  on  dialysis.  It  is  much 
less  soluble  than  serglobulin  in  an  excess  of  dilute  acetic  acid  and  coagulates 
at  65-69°  C.  The  difficulty  of  solution  in  acetic  acid  is  used  by  Pekelharing 
as  an  important  means  of  separating  the  compound  proteids  from  the  globulins. 

Serglobulins,  also  called  paraglohulin  (KiJHNE),  fibrinoplastic  substance 
(Alex.  Schmidt),  serum-casein  (Panum^),  occur  in  the  plasma,  serum, 
lymph,  transudates  and  exudates,  in  the  white  and  red  corpuscles,  and 
probably  in  many  animal  tissues  and  form-elements,  though  in  small  quan- 
tities.    They  are  also  found  in  the  urine  in  many  diseases. 

The  so-called  serglobulin  is  without  doubt  not  an  individual  substance, 
but  consists  of  a  mixture  of  two  or  more  protein  bodies  which  cannot  be 
completely  and  positively  separated  from  each  other.  The  mixture  of 
globulins  obtained  from  blood-plasma  or  blood-serum  by  saturation  with 
magnesium  sulphate  or  half-saturation  with  ammonium  sulphate  consists 
of  nucleoproteid,  fibrin-globulin,  and  the  true  serglobulin  or  mixture  of 
globulins. 

The  nucleoproteid  has  already  been  discussed.  The  fibrm-globulin, 
which  occurs  in  the  serum  only  in  small  amounts,  can  be  completel}'  pre- 
cipitated by  NaCl.  It  has  the  general  properties  of  the  globulins,  but 
differs  from  the  serglobulins  by  a  lower  coagidation  temperature,  64-66°  C, 
and  also  in  that  it  is  precipitated  by  (NH4)2S04  even  at  28  per  cent  satura- 
tion. 

Serglohvlins.  If  the  globulin  obtained  by  saturation  with  magnesium 
sulphate  is  dialyzed,  then,  as  has  been  known  for  a  long  time  and  further 
substantiated  by  Marcus,  only  a  part  of  the  globulin  separates  out,  while  a 
portion  remains  in  solution  and  cannot  be  precipitated  by  the  addition  of 
acid.  For  this  reason  iMarcus  2  also  differentiates  between  a  water-soluble 
globulin  and  one  insoluble  in  water.  According  to  the  recent  investigations 
of  HoFMEiSTER  and  PiCK^  the  part  insoluble  in  water  corresponds  chiefly 
to  a  globulin  fraction  readily  precipitated  by  (NH4)2S04  (by  28-36  vols. 

^  Kiihne,  Lehrbuch  d.  physiol.  Chem.,  Leipzig,  1866-68;    Alex.  Schmidt,  Arch,  f 
(Anat.  u.)  Physiol,   1861-62;    Panuni,  Virchow's  Arch.,  3  and  4. 
'  Zeitschr.  f.  physiol.  Chem.,  2S. 
^  Hofmeister's  Beitrage,  1. 


SERGLOBULIXS.  179 

per  cent  saturated  solution),  and  the  part  soluble  in  water  corresponds  to  a 
more  difficultly  precipitable  fraction  (by  36-44  vols,  per  cent  saturated  solu- 
tion). The  first  fraction  is  called  euglobulin  and  the  second  pseudoglobulin. 
According  to  Porges  and  Spiro  ^  the  serglobulins  can  be  separated  by 
(NH4)2S04  into  three  fractions  whose  precipitation  limits  are  28-36,  33-42, 
and  40-46  vols,  per  cent  saturated  solution.  All  three  fractions  contain 
globulin  insoluble  in  water.  Freund  and  Joachim  ^  have  recently  found 
that  the  euglobulin  as  well  as  the  pseudoglobulin  fraction  is  a  mixture  of 
globulin  soluble  in  water  and  globulin  insoluble  in  water,  and  consequently 
the  numlDer  of  different  globulins  in  the  ssrum  may  be  still  greater. 

It  follows  from  all  these  investigations  that  either  the  difference  between 
the  globulin  soluble  in  water  and  that  insoluble  is  not  sufficient  or  that  the  frac- 
tional precipitation  with  ammonium  sulphate  is  not  suited  for  the  separation  of 
the  various  globulins.  This  latter  seems  to  be  the  case,  as  shown  by  Haslam.^ 
It  must  not  be  forgotten  that  the  globulin  fractions  are  always  contaminated 
with  other  serum  constituents  and  that  these  may  influence  the  solubilities  and 
precipitability.  As  Hammarsten  has  shown,  a  water-soluble  globulin  can  be 
transformed  into  a  globulin  insoluble  in  water  by  careful  purification,  and  also 
the  reverse,  namely,  a  globulin  insoluble  in  water  can  sometimes  be  converted 
into  one  soluble  in  water  by  allowing  it  to  lie  in  the  air.  An  insoluble  protein 
ike  casein  can  also,  according  to  Hammarsten,"  have  the  solubilities  of  a  globulin 
due  to  contamination  with  constituents  of  the  serum,  and  K.  Mqrner  ^  has  also 
shown  that  a  contamination  of  the  serum-globulins  with  soap  can  essentiall}' modify 
the  precipitation  of  these  globulins.  Under  these  circumstances  the  above  state- 
ments in  regard  to  the  different  globulin  fractions  must  be  accepted  with  great 
caution. 

The  mvestigations  made  thus  far  upon  the  so-called  serglobulm  have 
not  led  to  any  positive  results.  That  this  globulin,  with  the  exception  of 
the  enzymes,  immune  bodies,  and  other  unknown  substances  which  are 
carried  douTi  by  the  various  fractions,  is  a  mixture  of  globulins  there  seems 
to  be  no  doubt.  The  serglobulin  or  the  globulin  mixture  which  is  obtained 
from  the  senmi  by  the  methods  to  be  described  has  the  following  properties. 

In  a  moist  condition  it  forms  snow-wliite  flaky  masses,  neither  tough 
nor  elastic,  which  always  contain  thrombin  and  hence  can  bring  about 
coagulation  in  a  fibrinogen  solution.  The  neutral  solution  is  only  incom- 
pletely precipitated  by  NaCl  added  to  saturation  and  is  not  precipitated  by 
an  equal  volume  of  a  saturated  salt  solution.  It  is  only  partly  precipitated 
by  dialysis  or  by  the  addition  of  acid.  On  saturation  with  magi:esium 
sulphate  or  one-half  saturation  vdth  ammonium  sulphate  a  complete  pre- 
cijiitation  is  obtained.  The  coagidation  temperature  is,  with.  5-10  per  cent 
NaCl  in  solution,  69-76°,  but  more  often  75°  C.     The  specific  rotation  of  the 


*  Hofmeister's  Beitrage,  3. 

'  Zeitschr.  f.  physiol.  Chern.,  36. 
^  Journ.  of  Physiol.,  32. 

*  See  Hammarsten,  Ergebnisse  d.  Physiol.,  1,  Abt.  1. 

*  Zeitschr.  f.  physiol.  Chem.,  34. 


180  THE  BLOOD. 

solution  containing  salt  is  (a)D=  —47.8°  for  the  serglobulin  from  ox-blood 
(FredericqI).  The  various  globulin  fractions  do  not  differ  essentially 
from  each  other  in  their  coagulation  temperatures,  specific  rotation,  refrac- 
tion coefficient  (Reiss^),  and  their  elementary  composition.  The  average 
composition  is,  according  to  Hammarsten,  C  52.71,  H  7.01,  N  15.85,  S  1.11 
per  cent.  K.  Morner^  found  1.02  per  cent  sulphur  and  0.67  per  cent  lead- 
blackening  sulphur.    All  the  sulphur  seems  to  exist  as  cystine. 

Serglobulin  contains,  as  K.  .Morner  first  showed,  a  carbohydrate  group 
which  can  be  split  off.  Langstein^  has  obtained  several  carbohydrates 
from  the  blood-globulin,  namely,  dextrose,  glucosamine,  and  carbohydrate 
acids  of  unknown  kinds.  It  has  not  been  shown  whether  these  small  amounts 
of  carbohydrate  are  derived  from  the  globulin  or  from  other  contaminating 
bodies.  According  to  Zanetti  the  blood-serum  contains  a  glucoproteid, 
and  the  investigations  of  Eichholz  ^  seem  to  show  that  the  globulins  are 
contammated  by  a  glucoproteid.  According  to  Langstein  the  sugar  is  not 
only  mixed  with  the  globulin,  but  it  exists  in  a  combined  form,  probably  in 
loose  combination. 

Serglobulm  (the  euglobulin)  may  be  easily  separated  as  a  fine  floc- 
culent  precipitate  from  blood-serum  by  neutralizing  or  making  faintly 
acid  with  acetic  acid  and  then  diluting  with  10-20  vols,  of  water.  For 
further  purification  this  precipitate  is  dissolved  in  dilute  common  salt 
solution,  or  in  water  by  the  aid  of  the  smallest  possible  amount  of  alkali, 
and  then  reprecipitated  by  diluting  with  water  or  by  the  addition  of  a 
little  acetic  acid.  All  the  serglobulin  may  also  be  separated  from  the 
serum  by  means  of  magnesium  or  ammonium  sulphate;  in  these  cases  it 
is  difficult  to  completely  remove  the  salt  by  dialysis.  As  long  as  we  are 
not  agreed  as  to  the  number  of  globulins  in  the  serum,  it  is  not  necessary 
to  give  a  method  of  separating  the  various  globulins  in  this  mixture.  Thus 
far  the  fractional  precipitation  with  (NH4)2S04  has  been  used  chiefly.  The 
serglobulin  from  blood-serum  is  always  contaminated  by  lecithin  and 
thrombin.  A  serglobulin  free  from  thrombin  may  l^e  prepared  from  fer- 
ment-free transudates,  as  sometimes  from  hydrocele  fluids,  and  this  shows 
that  serglobulin  and  thrombin  are  different  bodies.  For  the  detection 
and  the  quantitative  estimation  of  serglobulin  we  may  use  the  precipi- 
tation by  magnesium  sulphate  added  to  saturation  (Hammarsten),  or  by 
an  equal  volume  of  a  saturated  neutral  ammonium-sulphate  solution  (Hof- 
meister  and  Kauder  and  Pohl  ^).     In  the  quantitative  estimation  the 

'Bull.  Acad.  Roy.  de  Belg.  (2),  50.     In  regard  to  paraglobulin,  see  Hammarsten, 
Pfliiger's  Arch.,  17«and  18,  and  Ergebnisse  d.  Physiol.,  1,  Abt.  1, 
^  Hofmeister's  Beitrage,  4. 

*  Zeitschr.  f.  physiol.  Chem.,  34. 

*  Morner,  Centralbl.  f.  Physiol.,  7;  Langstein,  Miinch.  med.  Wochenschr.,  1902, 
1876,  and  Wien.  Sitzungsber.,  112,  Abt.  llh,  1903;  Monatsheft  f.  Chem., 25;  Hofmeister's 
Beitrage,  6;  see  also  foot-note  1,  p.  33. 

"  Zanetti,  Chem.  Centralbl.,  1898,  I,  p.  624;   Eichholz,  Journ.  of  Physiol.,  23. 
°  Hammarsten,  1.  c;   Hofmeister,  Kauder  and  Pohl.  Arch.  f.  exp.  Path.  u.  Pharm., 
20. 


SERALBUMINS.  181 

precipitate  is  collected  on  a  weighed  filter,  washed  with  the  salt  solution 
employed,  dried  with  the  filter  at  about  115°  C,  then  washed  with  boiling- 
hot  water,  so  as  to  completely  remove  the  salt,  extracted  with  alcohol  and 
ether,  dried,  weighed  and  incinerated  to  determine  the  ash.  The  accuracy 
of  these  methods  is  questionable,  as  shown  by  the  researches  of  Haslajvi. 

Seralbumins  are  found  in  large  quantities  in  blood-serum,  blood-plasma, 
lymph,  transudates,  and  exudates.  Probably  they  also  occur  in  other 
animal  fluids  and  tissues.  The  proteids  which  pass  into  the  urine  under 
pathological  conditions  consist  largely  of  seralbumin. 

The  seralbumin  like  the  serglobulin  seems  also  to  be  a  mixture  of  at 
least  two  proteid  bodies.  The  preparation  of  crystalline  seralbumin  (from 
horse-serum)  was  first  performed  by  Gurber.  It  crystallizes  with  difficulty 
from  other  blood-sera  (Gruzewska).  Even  from  horse-serum  only  a 
portion  of  the  albumins  is  obtained  as  crystals,  and  it  is  also  possible  that 
the  amorphous  albumin,  which  is  precipitated  by  ammonium  sulphate 
with  difficulty,  represents  two  seralbumins  (Maximowitsch).  According 
to  the  statements  of  Gl-rber  and  Michel  it  would  seem  that  the  cr>'s- 
talline  seralbumin  is  also  a  mixture,  but  this  is  disproved  by  the  obser- 
vations of  ScHULZ,  WiCHMAxx,  and  Krieger.^  We  know  nothing  as  to 
the  behavior  of  the  amorphous  fraction  of  the  seralbumin  in  this  regard. 
Because  of  the  different  coagulation  temperatures,  Halliburton  claims 
the  existence  of  three  different  albumins  in  the  blood-serum,  a  view  which 
has  been  disputed  by  several  experimenters  and  recently  by  Hougardy. 
On  the  other  hand,  the  older  investigations  of  Kauder,  as  well  as  the  more 
recent  work  of  Oppexheimer,^  seem  to  indicate  a  non-unit  nature  of  the 
seralbumins,  but  this  question  is  still  an  open  one. 

The  crystalline  seralbumin  may  perhaps  ])e  a  combination  with  sulphuric 
acid  (K.  MoRXER,  Ixagaki).  The  coagulated  albumin  obtained  from  the 
aqueous  solution  of  the  crystals  by  the  aid  of  alcohol  has  nearly  the  same 
elementary  composition  (Michel)  as  the  amorphous  mixture  of  albumin 
prepared  from  horse-serum  (Hammarstex  and  K.  Starke  ^).  The  average 
composition  was  C  53.06,  H  6.98,  N  15.99,  S  1.84  per  cent.  K.  Morxer, 
after  the  removal  of  the  sulphuric  acid  from  crystalline  albumin,  found 
1.73  per  cent  total  sulphur,  which  probably  exists  onh'  as  cystine.  Laxg- 
STEix  "*  has  been  able  to  split  off  a  nitrogenous  carl)ohydrate  (glucosamine) 
from  crystalline  serall)umin.     The  quantity  was  so  small  that  the  question 

'  In  regard  to  the  literature  on  the  crystalline  seralbumins,  see  Schulz,  Die  Kristal- 
lisation  von  Eiweissstoffen,  Jena,  1901;    Maximowitsch,  Maly's  Jahresber.,  31,  35. 

^  Halliburton,  Journ.  of  Physiol.,  5  and  7;  Hougardy,  Centrabl.  f.  Physiol.,  15, 
665;    Oppenheimer,  Verhandl.  d.  physidl.  Gesellsch.,  Berlin,  1902. 

'  Michel,  Verhandl.  d.  phys.-med.  Gesellsch.  zu  Wiirzburg,  29,  No.  3;  K.  Starke, 
Maly's  Jahresber  ,  11;    K.  Morner,  1.  c;    Inagaki,  Biochem.  Cenlralbl.,  4,  p.  515. 

*  K.  Morner,  1.  c;   Langstein,  Hofmeister's  Beitrage,  1. 


lo2  THE  BLOOD. 

is  still  undecided  whether  or  not  the  carbohydrate  was  not  a  contamination. 
The  fact  that  Abderhalden,  Bergell,  and  Dorpinghaus  ^  were  able  to 
prepare  a  seralbumin  entirely  free  from  carbohydrate  and  which  did  not 
respond  to  Molisch's  very  delicate  reaction  seems  to  be  decisive  on  this 
point.  The  specific  rotation  of  crystalline  seralbumins  from  horse-serum 
was  found  by  Michel  to  be  (a)D= -61-61.2°  and  by  Maximowitsch  on 
the  contrary  (a) d=  — 47.47°. 

The  crystalline  and  amorphous  seralbumin  in  aqueous  solution  give 
the  ordinary  albumin  reactions.  The  coagulation  temperature  of  a  1  per 
cent  solution  poor  in  salts  is  about  50°  C,  but  rises  with  the  quantity  of 
salt.  The  coagulation  of  the  mixture  of  albumins  from  serum  generally 
takes  place  at  70-85°  C,  but  is  essentially  dependent  upon  the  reaction  and 
the  amount  of  salt  present.  Up  to  the  present  time  no  seralbumin  solution 
has  been  prepared  free  from  mineral  bodies.  A  solution  as  free  from  salts 
as  possible  does  not  coagulate  either  on  boiling  or  on  the  addition  of  alco- 
hol.    On  the  addition  of  a  little  common  salt  it  coagulates  in  both  cases.2 

Seralbumin  differs  from  the  albumin  of  the  white  of  the  hen's  egg  in 
the  following  particulars:  It  is  more  levogyrate;  the  precipitate  formed  by 
hydrochloric  acid  easily  dissolves  in  an  excess  of  the  acid;  it  is  rendered 
less  insoluble  by  alcohol. 

In  preparing  the  seralbumin  mixture,  first  remove  the  globulins,  accord- 
ing to  JoHANSSOxN,  by  saturating  with  magnesium  sulphate  at  about  30°  C. 
and  filtering  at  the  same  temperature.  The  cooled  filtrate  is  separated 
from  the  crystallized  salt  and  is  treated  with  acetic  acid  so  that  it  contains 
about  1  per  cent.  The  precipitate  formed  is  filtered,  pressed,  dissolved 
in  water  with  the  addition  of  alkali  to  neutral  reaction  and  the  solution 
freed  from  salt  by  dialysis.  The  mixture  of  albumins  may  be  obtained 
in  a  solid  form  from  the  dialyzed  solution  either  by  evaporating  the  solu- 
tion at  a  gentle  temperature  or  by  precipitating  with  alcohol,  which  must 
be  quickly  removed.  Starke  ^  has  suggested  another  method,  which  is 
also  to  be  recommended.  The  crystalline  seralbumin  may  be  prepared 
from  serum  freed  from  globulin  ])y  half  saturating  with  ammonium  sul- 
phate, by  the  addition  of  more  salt  until  a  cloudiness  occurs,  and  then 
proceeding  according  to  the  suggestion  of  Gurber  and  Michel.  By 
acidification  with  acetic  acid  or  sulphuric  acid  the  crystallization  may 
be  considerably  enhanced.*  In  the  detection  and  quantitative  estimation 
of  seralbumin  the  filtrate  from  the  globulin  precijDitated  with  magnesium 
sulphate  can  be  heated  to  boiling,  after  acidification  with  a  little  acetic  acid 
if  necessary.  The  quantity  of  seralbumin  is  best  calculated  as  the  difference 
bet\\een  the  total  proteins  and  the  globulin. 

'  Zeitschr.  f.  physiol.  Chem.,  41. 

^  In  regard  to  the  relationship  of  neutral  saUs  to  heat  coagulation,  see  J.  Starke, 
;Sitzungsber.  d.  Gesellsch.  f,  Morph.  u.  Physiol,  in  Miinchen,  1897. 

^  Johansson,  Zeitschr.  f.  physiol.  Chem.,  9;    K.  Starke,  Maly's  Jahresber.,  11. 

^  See  Hopkins  and  Pinkus,  Journ.  of  Physiol.,  23;  Krieger,  tjber  die  Darstellung 
Icrj'stallinscher  tierischer  Eiweissstoffe,  Inaug.-Dissert.  Strassburg,  1899. 


BLOOD  SERUM.  183 

Summary  of  the  elementary  composition  of  the  above-mentioned  and  described 
proteins  (from  horse-blood) : 

Fibrinogen 52 . 93  6 . 90  16. 66  1 . 25         22 . 26  (Hammarstex) 

Fibrin 52.68  6. S3  16.91  1.10         22.48 

Fibrin-globulin 52  .  70  6 .  98  16 .  06  

Serglobulin 52.71  7.01  15.85  1.11         23.32               " 

Seralbumin 53.08  7.10  15.93  1.90         21 .96  (Michel) 

Embden  and  Knoop  as  well  as  Langstein  have  detected  in  blood-serum 
proteose-like  substances  which,  according  to  them,  occur  preformed  in  the  blood. 
KoLF  has  also  found  a  small  ciuantity  of  proteoses  in  the  blood  after  an  abundant 
absorption  of  proteoses  by  the  intestine.  According  to  Abderhaldex  and  Oppen- 
HEiMER  '  the  proteoses  cannot  be  considered  as  normal  blood  constituents;  even 
if  we  admit  of  their  presence,  the  cjuantity  is  too  small  to  be  of  any  physiological 
importance. 

v.  Bergmaxn  and  Langsteix  ^  have  determined  in  dogs  the  residual  nitrogen 
in  the  blood-serum,  i.e.,  the  nitrogen  of  the  non-coagulable  constituents.  They 
found  after  feeding  proteids  that  of  the  residual  nitrogen  25  per  cent  existed 
as  proteoses  and  5.5  per  cent  as  other  products  precipitable  with  phosphotung- 
stic  acid.  In  starving  animals  they  found  a  maximum  of  9  per  cent  of  the 
residual  nitrogen  as  proteoses. 

The  Blood-serum. 

As  above  stated,  the  blood-serum  is  the  clear  liquid  which  is  pressed  out 
by  the  contraction  of  the  blood-clot.  It  differs  chiefly  from  the  plasma  in 
the  absence  of  fibrinogen  and  in  containing  an  abundance  of  fibrin  ferment. 
Considered  qualitatively,  the  blood-serum  contains  the  same  chief  constitu- 
ents as  the  blood-plasma. 

Blood-senim  is  a  sticky  liquid  which  is  more  alkaline  towards  litmus 
than  the  plasma.  The  specific  gra\'ity  in  man  is  1.027  to  1.032,  average 
1.028.  The  color  is  often  strongly  or  faintly  yellow;  in  human  blood- 
serum  it  is  pale  yellow  ^^•ith  a  shade  towards  green,  and  in  horses  it  is  often 
amber-3^ellow.  The  serum  is  ordinarily  clear;  after  a  meal  it  may  be 
opalescent,  cloudy,  or  milky  white,  according  to  the  amount  of  fat  contained 
in  the  food. 

Besides  the  above-mentioned  bodies,  the  following  constituents  are 
found  in  the  blood-plasma  or  blood-serum: 

Fat  occurs  from  1-7  p.  m.  in  fasting  animals.  After  partaking  of  food 
the  amoimt  is  increased  to  a  great  extent.  Soaps,  cholesterin,  and  lecithin 
are  also  found.  Cholesterin  occurs,  according  to  HtJRTHLE,^  at  least  in 
part,  as  fatty-acid  esters  (seroh'yi  according  to  Boudet). 

'  Embden  and  Ivnoop,  Hofmeister's  Beitrage,  3;  Langstein,  ibid.,  3;  Nolf,  Bull. 
Acad.  Roy.  Belg.,  1903  and  1904;  Abderhalden  and  Oppenheimer,  Zeitschr.  f.  physiol. 
Chem.,42. 

^  V.  Bergmann  and  Lang.stein,  Hofmeister's  Beitrage,  6. 

^  Zeitschr.  f.  physiol.  Chem.,  21,  where  Boudet  is  also  cited.  In  regard  to  the 
Quantity  of  these  esters  in  bird-serum,  see  Brown,  Amer.  Journ.  of  Physiol.,  2. 


184  THE  BLOOD. 

Sugar  seems  to  be  a  physiological  constituent  of  the  plasma  and  serum. 
According  to  the  investigations  of  Abeles,  Ewald,  Kijlz,  v.  jMering, 
Pavy,  Seegen,  and  Miura^  the  sugar  found  is  dextrose.  Strauss  ^  has 
also  detected  levulose  in  blood-serum  and  in  transudates  and  exudates. 
The  question  as  to  the  occurrence  of  other  varieties  of  sugar,  such  as  iso- 
maltose  (Pavy  and  Siau)  and  pentose  (L:6pine  and  Boulud^),  in  blood- 
serum  is  still  undecided.  Asher  and  Rosenfeld*  have  shown  that  at 
least  a  considerable  part  of  the  sugar  can  be  removed  from  the  blood  by 
dialysis,  hence  it  must  exist  in  solution  in  the  free  state.  These  observa- 
tions do  not  exclude  the  possibility  of  the  existence  of  a  part  of  the  sugar 
in  a  combmed  form,  as  above  stated  (p.  180).  Besides  sugar  the  blood- 
serum  contains,  as  first  shown  by  J.  Otto,  also  another  reducing  non- 
fermentable  substance.  The  statements  of  Jacobsen,  Henriques,  and 
BiNG,^  that  this  substance  is  jecorin  or  lecithin  sugar,  do  not  have  sufficient 
foundation.  The  nature  of  another  carbohydrate  in  the  blood,  which  is 
neither  dextrorotatory  nor  reducing  and  which  has  been  called  virtual  sugar 
by  its  discoverers,  Lepine  and  Boulud,^  is  also  undetermined.  The  virtual 
sugar  is  more  abundant  in  the  blood  of  the  right  ventricle  than  m  the  arterial 
blood,  and  this  in  turn  is  richer  than  venous  blood.  In  the  passage  of  the 
blood  through  the  lungs  the  virtual  sugar  is  converted  into  ordinary  sugar; 
this  may  also  occur  in  the  capillaries  of  the  greater  circulatory  system. 

Conjugated  glucuronic  acids,  which  probably  originate  from  the  form- 
elements,  have  been  shown  to  occur  in  blood  by  the  researches  of  P.  ^Iayer, 
Lepine  and  Boulud.^  The  last  two  investigators  find  two  definite  glucu- 
ronic acids  in  the  blood,  both  of  which  are  levorotatory.  One  reduces 
Fehling's  solution  even  at  a  temperature  below  100°,  while  the  other 
reduces  it  at  above  100°.  Such  large  amounts  of  the  first  acid  often  occur 
in  the  blood  of  dogs  that  the  optical  activity  of  the  glucuronic  acid  coun- 
teracts that  of  the  glucose.  The  second  acid  also  occurs  in  larger  quantities 
as  compared  with  the  sugar. 

Bernard  ^  has  shown  that  the  quantity  of  sugar  in  the  blood  diminishes 

'  See  V.  Mering,  Arch.  f.  (Anat.  u.)  Physiol.,  1877  (this  article  contains  numer- 
ous references);   Se^gen,  Pfliiger's  Arch.,  40;   Miura,  Zeitschr.  f.  Biologie,  32. 

2  Fortschritte  d.  Mediz.,  1902. 

^  Pavy  and  Siau,  Journ.  of  Physiol.,  2G;  Lepine  et  Boulud,  Compt.  rend.,  133,  135, 
and  13G. 

■*  Centralbl.  f.  Physiol.,  19,  p.  449. 

^Otto,  Pfliiger's  Arch.,  35  (a  good  review  of  the  older  literature  on  sugar  in  the 
blood);  Jacobsen,  Centralbl.  f.  Physiol.,  6  368;  Henriques,  Zeitschr.  f.  physiol.  Chem., 
23;   Bing,  Skand.  Arch.  f.  Physiol.,  9. 

"Compt.  rend.,  137. 

'  Mayer,  Zeitschr.  f.  physiol.  Chem.,  32;  Lepine  and  Boulud,  Compt.  rend.,  133, 
135, 136, 138, 141,  and  Journ.  de  Physiol.,  7  (cited  from  Biochem.  Centralbl.,  4,  p.  421). 

*  Lemons  sur  le  diabete,  Paris,  1877. 


BLOOD  SERUM.  185 

more  or  less  rapidly  on  lea\'ing  the  veins.  L:fipiNE,  associated  ^ith  Baeral^ 
has  specially  studied  this  decrease  in  the  quantity  of  sugar  and  calls  it 
glycolysis.  Lepine  and  Barral,  as  well  as  Arthus,  have  sho-wn  that  this 
glycolysis  takes  place  in  the  complete  absence  of  micro-organisms.  It 
seems  to  be  due  to  a  soluble  glycolytic  enzyme  whose  acti\-ity  is  destroyed 
by  heating  to  54°  C.  This  enzyme  is  derived,  according  to  the  above 
investigators,  from  the  leucocytes  and,  according  to  Lepine,^  has  some 
connection  ^\-ith  the  pancreas.  The  glycolysis  is,  according  to  Nasse, 
RoHMANN  and  Spitzer  and  Sieber,^  an  oxidation  wliich  is  produced,, 
according  to  the  two  last -mentioned  investigators,  by  an  oxidation  ferment. 
It  is  certainly  not  connected  with  the  sur\'ival  of  the  cells,  but  whether  it 
is  a  \-ital  or  a  post-mortem  process  is  not  decided.'^ 

The  blood-plasma  and  the  serum,  as  well  as  the  lymph,  also  contain 
enzymes  of  various  kinds.  According  to  Roh^l\xx,  Bl\l,  Hamburger,* 
and  others,  diastases,  which  convert  starch  and  glycogen  into  maltose  or 
isomaltose,  as  well  as  a  maltoglucase  are  found  in  the  blood.  Hanriot 
has  detected  a  lipase  in  the  senmi  which  decomposes  butyrin,  and  which, 
according  to  him,  decomposes  neutral  fats  and  other  esters.  The  occur- 
rence of  a  hutyrinase  is  generally  admitted,  while  the  property  of  this  lipase 
of  splitting  olein  and  other  neutral  fats  is  not  generally  acknowledged 
(Arthus,  Doyox  and  ]\1orel^).  Thi«  lipolytic  property,  if  it  exists  to 
the  extent  that  Hanriot  ascribes  to  it,  must  not  be  confounded  with  the 
transformation  of  fat  into  unknown  substances  soluble  in  water,  a  phenom- 
enon first  observed  by  Cohxsteix  and  ^Iichaelis  and  further  studied  by 
Weigert.^  This  property  seems  to  be  connected  with  the  form-elements 
of  the  blood. 

Besides  the  above-mentioned  enzymes  and  thrombin,  several  other 
enzymes  have  been  found  in  the  iDlood-serum,  namely,  oxidases,  catalases, 

'  I'l  regard  to  the  numerous  memoirs  of  Lepine  and  Lepine  et  Barral,  see  Lyon 
medical.,  62  and  63;  Compt.  rendus,  110,  112,  113,  120,  and  139;  Lepine,  Le  ferment 
glycolytique  et  la  pathogenie  du  diabete  (Paris,  1891),  and  Revue  analytique  et 
critique  des  travaux,  etc.,  in  Arch,  de  med.  exper.  (Paris,  1892);  Revue  de  medecine, 
1895;  Arthus,  Arch,  de  Physiol.  (5),  3,  4;  Xasse  and  Framm,  Pfiuger's  Arch.,  63, 
Paderi,  Maly's  Jahresber.,  26;  see  also  Cremer,  Physiologic  des  Glykogens  in  Ergebnisse 
d.  Physiol.,  1,  Abt.  1. 

^  See  Chapter  I  and  N.  Sieber,  Zeitschr.  f.  physiol  Chem.,  39  and  44. 

^  See  Arthus,  1.  c;  Colenbrander,  Maly's  Jaliresber.,  22;  RjTvosch,  Centralbl.  f. 
Physiol.,  11,  495. 

^  Rohmann;  Rohmann  and  Hamburger,  Ber.  d.  deutsch.  chem.  Gesellsch.,  25  and 
27;  Pfli'iger's  Arch.,  52  and  60;  Bial,  Leber  das  diast.  Perm.,  etc.,  Inaug.-Diss.  Breslau, 
1892  (older  hterature).     See  also  Pfliiger's  Arch.,  52,  54.  and  55. 

^Hanriot,  Compt.  rend.  soc.  biol.,  48  and  54;  Compt.  rend.,  123  and  132;  Arthus, 
Journ.  de  Physiol,  et  de  Pathol.,  4;  Doyon  and  Morel,  Compt.  rend.  toe.  biol.,  54; 
Achard  and  Clerr  (Lipase  in  Eisease),  Compt.  rend.,  129,  and  Arch.  d.  med.  exper.,  14. 

*  Cohnstein  and  Michaelis,  Pfliiger's  Arch.,  65  and  69;  Weigert,  ibid.,  82. 


186  THE  BLOOD. 

proteolytic  enzymes,  rennin,  and  trypsin,  and  also  the  corresponding  anti- 
enzymes.  We  cannot  enter  into  the  discussion  of  these,  nor  of  the  many 
not  chemically  characterized  bodies  which  have  been  called  toxines  and 
antitoxines,  immune  bodies,  alexines,  hcemolysines,  cytotoxines,  etc.  It  is  also 
not  within  the  scope  of  this  book  to  discuss  the  jwecipitines  which  can  be 
used  as  a  biological  reagent  on  account  of  their  action  upon  various  pro- 
teins. It  may  be  sufficient  to  state  that  the  works  of  Bordet,  Ehrlich, 
Wassermann,  Schutze,  Uhlenhaut/  and  others  have  shown  that  the 
repeated  injection  into  an  animal  of  a  foreign  protein  body  or  of  blood  of  a 
different  species  of  animal  so  changes  the  blood  of  this  animal  that  it  acquires 
precipitating  properties  towards  the  injected  protein  or  blood.  In  this 
manner  we  obtain  a  biological  reagent  for  various  proteins  and  for  blood  of 
different  animals.  This  last  behavior  has  become  of  great  forensic  impor- 
tance, due  to  the  work  of  Uhlenhaut.  The  various  enzymes  and  anti- 
enzymes,  toxines  and  antitoxines,  precipitines,  etc.,  are  as  a  rule  precipitated 
with  the  globulin,  but  differ  among  each  other  in  that  some  are  carried 
down  by  the  euglobulin,  while  the  others  are  carried  down  by  the  pseudo- 
globulin  fraction. 

Among  the  bodies  which  are  found  in  the  blood,  and  without  doubt  are 
met  with  in  smaller  or  greater  amounts  in  the  plasma,  are  to  be  mentioned 
urea,  uric  acid  (found  in  human  blood  by  Abeles),  jihosphocarnic  acid  (Pa- 
NELLA^)^  creatine,  carhamic  acid,  paralactic  acid,  hippuric  acid,  and  traces  of 
indol  (Hervieux^).  Under  pathological  conditions  the  following  bodies 
have  been  found:  xanthine  bodies,  leucine,  tyrosine,  lysine  (Neuberg  and 
RiCHTER  ^),  and  biliary  constituents. 

The  coloring-matters  of  the  blood-serum  are  very  little  known.  In 
equine  blood-serum  the  biliary  coloring-matter,  bilirubin,  besides  other  color- 
ing-matters, often  occurs.  The  yellow  coloring-matter  of  the  serum  seems 
to  belong  to  the  group  of  luteins,  which. are  often  called  lipochromes  or  fat- 
coloring  matters.  From  ox-serum  Krukenberg  ^  was  able  to  isolate  with 
amyl  alcohol  a  so-called  lipochrome  whose  solution  shows  two  absorption - 
bands,  of  which  one  encloses  the  line  F  and  the  other  lies  between  F  and  G. 

The  mineral  bodies  in  serum  and  plasma  are  qualitatively,  but  not 
quantitatively,  the  same.  A  part  of  the  calcium,  magnesium,  and  phos- 
phoric acid  is  removed  on  the  coagulation  of  the  fibrin.  -  By  means  of 
dialysis,  the  presence  of  sodium  chloride,  which  forms  the  chief  mass  or 

'  The  literature  on  this  subject  may  be  found  in  bacteriological  journals  and  works. 
See  also  L.  Miciiaelis,  Biochem.  Centralbl.,  3,  p.  693. 

'  Abeles,  Wien.  med.  Jahrb.,  1887;  Panella  cited  from  Virchow's  Jahresber.  f.  1902, 
150. 

'  Compt.  rend.  .'oc.  biolog.,  56. 

*  Deutsch.  n;ed.  Wochenschr.,  1904. 

*  Sitzungsber.  d.  Jen,  Gesellsch.  f.  Med.,  1885. 


MINERAL  CONSTITUENTS  OF  THE  SERUM.  1S7 

60-70  per  cent  of  the  total  mineral  bodies,  lime-salts,  sodium  carbonate, 
and  traces  of  sulphuric  and  phosphoric  acids  and  of  potassium,  may  be 
directly  shown  in  the  serum.^  Traces  of  silicic  acid,  fluorine,  copper,  iron, 
manganese,  and  ammonia  are  claimed  to  have  been  found  in  the  serum. 
As  in  most  animal  fluids,  the  chlorine  and  sodium  are  in  the  blood-serum  in 
excess  of  the  phosphoric  acid  and  potassium  (the  occurrence  of  which  in 
the  serum  is  even  doubted).  The  acids  present  in  the  ash  are  not  sufficient 
to  saturate  the  bases  found,  a  condition  which  shows  that  a  part  of  the 
bases  is  combined  with  organic  substances,  perhaps  proteins.  This  coin- 
cides also  with  the  fact  that  the  great  part  of  the  alkalies  does  not  exist 
in  the  senmi  as  diffusible  alkali  compounds,  carbonate  and  phosphate,  but 
as  non-diffusible  compounds,  protein  combinations.  According  to  Ham- 
burger ^  37  per  cent  of  the  alkali  of  the  serum  from  horse-blood  was  dif- 
fusible and  63  per  cent  non-diffusible. 

Iodine,  which  seems  to  be  habitually  found,  is  also  considered  as  a 
mineral  constituent  of  the  plasma  or  serum  (Gley  and  Bourcet),  while 
arsenic,  which  is  not  found  in  all  blood  occurs  only  in  human  blood 
(Gautier,  Bourcet 3).  Iodine  occurs  to  a  greater  extent  in  menstnial 
blood  than  in  other  blood  and  does  not  exist  as  a  salt,  but  as  an  organic 
compound  (Bourcet). 

The  gases  of  the  blood-serum,  which  consist  chiefly  of  carbon  dioxide 
with  only  a  little  nitrogen  and  oxygen,  will  be  described  when  treating  of 
the  gases  of  the  blood. 

Because  of  the  difficulty  of  obtaining  plasma  only  a  few  analyses  have 
been  made.  As  an  example  the  results  of  the  analyses  of  the  blood-plasma 
of  the  horse  will  be  given  below.  The  analysis  No.  1  was  made  by  Hoppe- 
Seyler.4  ]n^Tq_  2  is  the  average  of  the  results  of  three  analyses  made  by 
Hammarstex.     The  figures  are  given  for  1000  parts  of  the  plasma. 

No.  1.  No.  2. 

Water 908.4  917.6 

Solids 91.6  82.4 

Total  proteins 77.6  69.5 

Fibrin.     10.1  6.5 

Globulin. 38.4 

Seralbumin 24 . 6 

Fat 1.2 

Extractive  substances 4.0 


Soluble  salts 6.4  1  ^^'^ 

Insoluble  salts 1 . 7  J 

Lewinsky  ^  has  determined  the  total  proteins  and  the  individual  pro- 
teins in  the  blood-plasma  of  man  and  animals  with  the  following  results. 

'  See  Giirber,  Verhandl.  d.  phys.-med.  Gesellsch.  zu  Wiirzburg,  23. 

^  In  regard  to  method,  see  Arch.  f.  (Anat.  u.)  Physiol.,  1898. 

^  Gley  et  Bourcet,  Compt.  rend.,  130;   Bourcet,  ibid.,  131;   Gautier,  ibid.,  131. 

*  Cit.  from  v.  Gorup-Besanez's  Lehrbuch  der  physiol.  Chem.,  4.  Aufl.,  346. 

^Pfliiger's  Arch.,   100. 


188  THE  BLOOD. 

Total  Protein.  Albumin.  Globolin.  Fibrinogen. 

Man 72.6  40.1  28.3            4.2 

Dog 60.3  31.7  22.6             6.0 

Sheep 72.9  38.3  30.0             4.6 

Horse 80.4  28.0  47.9             4.5 

Pig SO. 5  44.2  29.8            6.5 

Abderhaldex  has  made  complete  analyses  of  the  blood-.semm  of  several 
domestic  animals.  From  the.=e  analyses  as  well  as  from  those  made  by 
Hammarstex  of  the  serum  from  htmian,  horse,  and  ox  blood  it  follows  that 
the  amount  of  solids  ordinarily  varies  betw-een  70-97  p.  m.  The  chief  mass 
of  the  solids  consists  of  proteins,  about  55-S4  p.  m.  In  hens  Hamm.ui- 
STEN  found  much  lower  values,  namely.  54  p.  m.  solids,  with  only  39.5  p.  m. 
protein,  and  Halliburtox  found  only  25.4  p.  m.  protein  in  frog's  bl<x>d. 
The  relationship  between  globulin  and  seralbumin  is.  as  shown  by  the 
analyses  of  H.odjarstex,  H.u.libuhtox,  and  Rubbrecht.i  very  different 
for  various  animals,  but  may  also  vsiry  considerably  in  the  same  species  of 
animal.  In  human  blood-serum  HAinL^JBXEx  found  more  seralbumin 
than  globulin,  and  the  relationship  of  sei^lobulin  to  seralbumin  was  as  1 :1.'5 
Lewixskt  foimd  the  relationship  in  man  greater  than  1.  indeed  1 : 1.39-2.13. 
In  regard  to  the  quantity  of  the  remaining  organic  constituents  of  the  serum 
we  refer  the  reader  to  Abderhaldex's  complete  analyses. 

In  starvation  it  .seems,  as  first  found  by  Brr.CKH.^RDT  and  recently  sub- 
stantiated by  GiTHExs.2  that  the  quantity  of  globulin  relative  to  that  of 
albumin  is  mcreased.  A  change  in  the  relationship  with  a  decrease  in  the 
albumin  and  increase  in  the  globulin  may  also  occur  in  animals  which  have 
been  made  sick  or  in  part  immune  by  inoculation  with  pathogenic  micro- 
organisms (Laxgsteix  and  ^L\yee3).  The  total  protein  content  is  raised 
in  nearly  all  cases.  The  amount  of  fibrinogen  in  the  plasma  is  espe- 
cially increased  by  pneumococci,  streptococci,  and  pas^taphylococci 
(P.  Muller^). 

The  quantity  of  nuneral  bodies  in  the  .serum  has  been  determined  by 
manv  investigators.  The  conclusion  drawn  from  the  analy.ses  is  that  there 
exists  a  rather  close  correspondence  between  htmian  and  animal  blood- 
serum,  and  it  Ls  therefore  sufficient  to  give  here  the  analysis  of  C.  Schmidt  * 
of  (1)  htmian  blood,  and  Buxge  and  Abderh-axdex's  anah-.ses  (2)  of  senun 
of  ox,  bull,  sheep,  goat,  pig,  rabbit,  dq?,  and  cat.  The  results  correspond 
to  1000  parts  by  weight  of  the  senun. 


'  Abderhalden.  Zeitschr.  f.  physiol.  Chem..  25;  Hammarsten.  Pfluger's  Arch.,  17; 
Halliburton.  Jo\im.  of  Physiol.,  7;  Rubbrecht,  Travaux  du  laboratoire  de  I'institut 
de  physiologie  de  Liege,  5,  1S96. 

2  Burckhardt.  Arch.  f.  exp.  Path.  u.  Pharm.,  16;  Githens,  Hofmeister's  Beitrage,  5. 

'  Hofmeister's  Beitrage,  5. 

*Ibid.,  6. 

scit.  from  Hoppe-Seyler.  Physiol.  Chem.,  T  SI,  p.  439. 


MINERAL  CONSTITUENTS  OF  THE  SERUM.  ISO 

1  2 

KoO 0.387-0.401  0.226-0.270 

Na,0 4.290-4.290  4.251-4.442 

CI 3.565-3.659  3.627-4.170 

CaO 0.155-0.155  0.119-0.131 

MgO 0.101 0.040-0.046 

P2O5  (inorg.) 0 . 052-0 . 0S5 

Even  if  we  bear  in  mind  that  certain  bodies,  such  as  carbon  dioxide, 
are  driven  off  durmg  uicmeration  and  that  other  bodies,  such  as  sulphuric 
acid  and  phosphoric  acid,  are  formed  from  sulphurized  and  phosphorized 
organic  substances,  still  quantitative  analyses  like  the  above  are  not 
sufficient  for  the  scientific  demands  of  to-day.  They  do  not  show  the 
true  composition  and  especially  do  not  give  an  explanation  of  the 
number  of  different  ions  present  m  the  serum  or  in  other  fluids,  a  question 
which  is  of  the  greatest  physiological  importance.  .\n  answer  to  these 
questions  is  obtainable  only  by  physico-chemical  investigations,  which  have 
thus  far  been  used  cliiefly  m  determming  the  molecular  concentration,  the 
amount  of  electrolytes  and  non-electrolytes,  and  the  degree  of  dissociation. 

The  molecular,  or,  as  Hamburger  calls  it,  the  osmotic  concentration  which  gives 
the  total  number  of  molecules  and  ions  in  the  litre,  is  measured  bv  the  osmotic 

J 

pressure,  and  it  may  be  expressed  bj'  7-^  if  we  make  use  of  the  depression  of  the 

freezing-point  (J)  instead  of  the  osmotic  pressure,  as  a  gram-molecule  of  a  non- 
electrolyte,  or  an  equivalent  number  of  ions,  when  in  1  litre  of  solution,  causes  a 
depression  of  the  freezing-point  of  1 .85°. 

The  average  depression  of  the  freezing-point  of  human  blood-serum  is 
ordinarily  given  as  J  =  —  0.526°.  According  to  Th.  CohxI  the  actual 
depression  of  the  freezing-pomt  of  normal  human  blood  is  J  =—0.537°. 
This  freezing-point  depression,  it  seems,  is  a  little  lower  than  that  of  the 
sera  of  other  mammals  that  have  been  investigated:  —0.560°  (horse)  to 
0.619°  (sheep).  The  molecular  concentration  of  the  blood-serum  of  various 
mammals  also  differs  only  slightly  in  each  case,  according  to  Bugarsky  and 
Taxgl.-  and  amounts  on  an  average  to  about  0.320  mol  per  litre.  The 
average  freezing-point  depression  corresponds  closely  to  that  of  a  common 
salt  solution  of  9  p.  m.  (J=— 0.551°  to  —0.561°).  and  at  present  such  a 
solution  is  considered  as  a  physiological  salt  solution  for  man  and  other 
mammals. 

The  conditions  are  otherwise  with  sea-animals  which  live  in  a  medium 
rich  in  salts.  According  to  Bottazzi  the  blood  (or  the  fluid  of  the  cavities) 
of  invertebrate  sea-animals  has  an  osmotic  pressure  which  corresponds  to 
an  average  freezing-point  depression  of  J  =  —  2.29°,  i.e..  exactly  the  same 

^  Mitteil.  aus  d.  Grenzgeb.  d.  Mediz.  u.  Chir..  1.'). 

■  In  regard  to  the  literature  on  this  subject  we  refer  to  Hamburger,  Osmotischer 
Druck  und  lonenlehre.  from  which  the  author  obtained  most  of  the  facts  given.  See 
also  Hober,  Physikalische  Chemie  der  Zelle  und  der  Gewebe,  2.  Aufl.,  1906. 


190  THE  BLOOD. 

as  the  sea-water  in  which  they  Uve.  In  tlie  cartilaginous  fishes  nearly  the 
same  conditions  exist,  while  in  the  Teleostei  the  osmotic  pressure  is  much 
lower  than  that  of  the  sea-water,  but  is  about  one  half  greater  than  the 
blood  of  land-vertebrates.  The  Teleostei  are  the  first  in  the  scale  of  de- 
velopment of  animals  to  show  an  independence  of  the  osmotic  pressure  of 
the  inner  fluids  from  the  surrounding  media. 

The  researches  of  Fredericq  ^  have  led  to  the  same  results.  In  the 
sea-invertebrates  examined  the  blood  (the  hsemolymph)  had  the  same 
molecular  concentration  and  same  salt  content  as  the  exterior  medium. 
In  plagiostoma  the  blood  had,  with  equal  molecular  concentration,  a  con- 
siderably lower  salt  content  than  the  sea-water.  The  equality  of  the 
molecular  concentration  was  maintained  in  these  cases  by  a  high  urea 
content.  In  all  bony  fishes  of  salt  and  fresh  waters  and  in  fresh-water 
crabs  the  blood  differs  markedly  in  regard  to  molecular  concentration,  as 
well  as  in  salt  content,  from  the  exterior  medium. 

There  are  recorded  a  great  number  of  investigations  on  the  changes  in 
the  osmotic  pressure  or  the  molecular  concentration  of  the  blood-serum 
under  various  physiological  conditions  as  well  as  in  disease,  but  still  it  is  no 
doubt  too  early  to  draw  any  definite  conclusions  from  these  observations. 

As  seen  from  the  above,  blood-serum  contains  electrolytes  as  well  as 
non-electrolytes.  Of  the  latter  the  proteins  and  also  sugar,  fat,  lecithin, 
urea,  and  the  so-called  extracti\e  bodies  are  of  the  greatest  importance. 
The  electrolytes  comprise  the  various  ions  and  the  undissociated  molecules 
of  the  salts  of  the  serum.  The  electrolytes  are  the  only  constituents  of 
the  serum  which  conduct  the  electric  current,  while  the  non-electrolytes 
retard  the  conductivity.  The  degree  of  dissociation  can  also  be  calculated 
from  the  determination  of  the  conductivity  of  the  blood-serum. 

The  coefficient  of  dissociation  is,  according  to  Arrhenius,  the  relationship 
between  the  number  of  ions  in  a  solution  and  the  number  of  ions  which  would 
be  present  if  the  electrolytes  were  completely  dissociated.  As  the  conductivity 
of  a  solution  of  electrolytes  is  determined  by  the  number  of  ions  (admitting  that 
the  migration  velocity  of  the  ions  is  the  same  for  different  dilutions),  the  above 

coefficient  a  can  be  calculated  by  the  formula  n  =  - — .     In  this  formula  h  repre- 

sents  the  conductivity  of  the  original  dilution  (i.e.,  of  the  undiluted  serum)  and 
^-jc  the  conductivity  of  the  completely  dissociated  molecules  (ions)  after  suffi- 
ciently strong  dilution  of  the  serum  with  water. 

According  to  the  above  principle  the  degree  of  dissociation  of  serum  has 
been  determined  by  several  investigators,  especially  Bugarsky  and  Tangl, 
Oker-Blom,  and  Viola.  This  last  .investigator  found  that  the  degree  of 
dissociation  of  the  blood-senim  of  healthy  human  beings  was  equal  to 
0.68-0.73.      According  to   Hamburger  the  results  thus  obtained  experi- 

'  Aich.  de  Biol.,  20.     Cited  from  Centialbl.  f.  Physiol.,  19.  p.  21. 


PHYSICO-CHEMICAL  PROPERTIES.  191 

mentally  must  be  a  little  too  low  for  certain  reasons,  and  we  therefore  can 
consider  the  dissociation  coefficient  to  be  between  0.65  and  0.82. 

As  above  stated,  the  non-electrolytes  have  a  retarding  action  upon  the  con- 
ductivity, and  according  to  Bugarsky  and  Tangl  each  gram  of  protein  in  100 
c.c.  of  serum  diminishes  the  electrical  conductivity  of  the  serum  about  2.5  per 
cent.  By  making  use  of  this  fact,  the  corrected  conductivity  of  the  electrolytes 
present  can  be  determined  from  the  conductivity.  The  corrected  conductivity 
is  partly  dependent  upon  the  chlorides  and  partly  upon  the  other  salts  (which  are 
nearly  identical  with  the  quantity  of  NaaCOg).  If  the  amount  of  NaCl  of  the 
serum  is  determined  by  analysis  we  can  calculate  the  conductivity  of  the  other  salts 
by  subtracting  the  calculated  conductivity  of  a  solution  of  NaCl  of  similar  con- 
centration (which  can  be  done  according  to  Kohlrausch's  method)  from  the  total 
corrected  conductivity.  From  these  results  we  can  calculate  the  molecular 
concentration  of  the  chlorides  and  of  the  non-chlorides.  The  sum  of  these  two 
is  subtracted  from  the  molecular  concentration  of  the  serum,  when  the  molecular 
concentration  of  the  non-electrolytes  is  obtained. 

Bugarsky  and  Tangl  have  made  physico-chemical  analyses  of  blood- 
serum  of  certain  mammals  according  to  the  principle  given  above.  They 
found  that  the  molecular  concentration  was,  on  an  average,  about  0.320 
mol  per  litre,  that  about  three  fourths  of  the  total  dissolved  molecules 
of  blood-serum  were  electrolytes,  although  the  serum  contained  about 
70-80  p.  m.  proteid  and  10  p.  m.  inorganic  bodies,  and  also  that  three 
fourths  of  the  quantity  of  electrolytes  consisted  of  NaCl.  Viola  and  Bous- 
QUET  have  recorded  less  complete  osmotic  chemical  analyses  of  blood-serum 
of  diseased  and  healthy  human  beings,  making  use  of  methods  somewhat 
different  in  principle. 

In  the  determination  of  the  alkalinity  of  blood  and  blood-serum,  up  to 
the  present  time  we  have  estimated  the  amount  of  alkali  by  titration  with 
an  acid.  We  cannot  dispense  with  such  determinations,  although  they  do 
not  yield  any  information  as  to  the  true  alkalinity,  apart  from  the  fact  that 
the  results  are  dependent  upon  the  indicator  used,  because  we  understand 
as  true  alkalinity  the  concentration  of  the  hydroxyl  ions.  The  Na2C03 
is  in  aqueous  solution  more  or  less  dissociated  into  2Na+  and  COs"",  depend- 
ing upon  the  dilution.  The  C03=  ions  combine  partly  with  the  H+  ions 
of  the  dissociated  water,  forming  HCOs",  and  the  corresponding  H0~  ions 
produce  the  alkaline  reaction.  If  now,  by  the  addition  of  a  little  acid,  a 
few  of  the  H0~  ions  are  removed,  then  the  equilibrium  is  disturbed,  a  new 
quantity  of  Na2C03  is  dissociated,  and  this  process  is  repeated  ever}'  time 
a  new  quantity  of  acid  is  added  until  all  the  carbonate  is  dissociated.  The 
dissociation  of  the  carbonate  existing  in  the  original  concentration,  upon 
which  the  number  of  HO~ions  is  dependent,  cannot  therefore  be  determined 
by  titration.  For  these  reasons  Hober  has  worked  out  a  physico-chemical 
method  of  determining  alkalinity,  based  upon  Nernst's  theoiy  of  liquid 
chains.  This  method  was  'used  later  by  Farkas,  Franckel,  and  Hober 
after  a  few  changes.     The  investigations  of  these  last -mentioned  experi- 


192  THE  BLOOD. 

menters  show  that  the  concentration  of  the  hydroxy!  ions  in  blood-serum 
and  blood  is  nearly  the  same  as  in  distilled  water,  and  that  these  fluids  are 
nearly  neutral  in  behavior,  which  fact  ic  caused  by  the  presence  of  carbonic 
acid.  Friedenthal,!  by  testing  serum  with  phenolphthalein,  arrived  at 
similar  results. 

n.   THE  FORM-ELEMENTS  OF  THE  BLOOD. 
The  Red  Blood-Corpuscles. 

The  blood-corpuscles  are  round,  biconcave  disks  without  membrane  and 
nucleus  in  man  and  mammalia  (with  the  exception  of  the  llama,  the  camel, 
and  their  congeners).  In  the  latter  animals,  as  also  in  birds,  amphibia,  and 
fishes  (with  the  exception  of  the  Cyclostoma),  the  corpuscles  have  in  general 
a  nucleus,  are  biconvex  and  more  or  less  elliptical.  The  size  varies  in 
different  animals.  In  man  they  have  an  average  diameter  of  7  to  8  ^ 
(/i= 0.001  mm.)  and  a  maximum  thickness  of  1.9  //.  They  are  hea\'ier 
than  the  blood-plasma  or  sennn,  and  therefore  sink  in  these  liquids.  In 
the  discharged  blood  they  may  lie  sometimes  with  their  flat  surfaces  to- 
gether, forming  a  cylinder  like  a  roll  of  coin  (rouleaux).  The  reason  for 
this  phenomenon,  which  is  considered  as  an  agglutination,  has  not  been 
suffic-'^'ntly  studied,  but  as  it  may  be  observed  in  defibrinated  blood  it  seems 
proba.  le  that  the  formation  of  fibrin  has  nothing  to  do  with  it. 

The  number  of  red  blood-corpuscles  is  different  in  the  blood  of  various 
animals.  In  the  blood  of  man  there  are  generally  5  million  red  corpuscles 
in  1  c.mm.,  and  in  woman  4  to  4.5  million. 

The  blood-corpuscles  consist  essentially  of  two  chief  constituents,  the 
stroma,  which  forms  the  real  protoplasm,  and  the  intraglobular  contents, 
whose  chief  constituent  is  haemoglobin.  We  cannot  state  anything  posi- 
tive for  the  present  in  regard  to  a  more  detailed  arrangement,  and  the  views 
on  this  subject  are  somewhat  divergent.  The  two  following  views  are 
more  or  less  related  to  each  other.  According  to  one  view  the  blood- 
corpuscles  consist  of  a  membrane  which  encloses  a  hsemoglo]:)in  solution, 
while  the  other  view  considers  the  stroma  as  a  protoplasmic  structure 
soaked  with  haemoglobin.  This  latter  view  is  in  accord  with  the  assump- 
tion as  to  an  outside  boundary-layer. 

Thus  according  to  Hamburger  the  stroma  forms  a  protoplasmic  net 
in  whose  meshes  there  exists  a  red  fluid  or  semi-fluid  mass  which  consists 
in  great  measure  of  haemoglobin.  This  mass  represents  the  water-attract- 
ing force  of  the  blood-corpuscles,  and  besides  this  it  is  also  considered  that 
the  outer  protoplasmic  boundary  is  semi-permeable,  i.e.,  permeable  to 
water  but  not  permeable  to  certain  crystalloids.     The  researches  of  Koppe, 

'  Hober,  PfliJger's  Arch.,  81  and  99;  Farkas,  see  Biochem.  Centralbl.,  1,  626; 
Franckel,  Pfliiger's  Arch.,  9fi;  Friedenthal,  Zeitschr.  f.  allg.  Physiol.,  1  and  4. 


THE  RED  CORPUSCLES.  193 

Albrecht,  Pascucci,  Rywosch,^  and  others  indicate  the  presence  of  a 
special  envelope  or  boundary-layer,  and  there  is  no  doubt  that  the  outer 
layer  contains  so-called  lipoids,  such  as  cholesterin,  lecithin,  and  similar 
bodies. 

The  red  blood-corpuscles  retain  their  volume  in  a  salt  solution  which 
has  the  same  osmotic  pressure  as  the  serum  of  the  same  blood,  although 
they  may  change  their  form  in  such  solutions,  becoming  more  spherical, 
and  may  also  midergo  a  chemical  change  (Hamburger,  Hedin,  and  others). 
Such  a  salt  solution  is  isotonic^  with  the  blood-serum,  and  its  concentra- 
tion for  a  NaCl  solution  is  approximately  9  p.  m.  for  human  and  mam- 
malian blood.  A  solution  of  greater  concentration,  a  hyperisotonic  solu- 
tion, abstracts  water  from  the  blood-corpuscles  imtil  osmotic  equilibrium 
is  established,  hence  the  corpuscles  shrink  and  their  volume  becomes 
smaller.  In  solutions  of  less  concentration,  hypisotonic  solutions,  the  cor- 
puscles swell  up,  due  to  the  taking  up  of  water,  and  this  swelling  may  be 
so  great,  as  on  diluting  the  blood  with  water,  that  the  haemoglobin  is  sepa- 
rated from  the  stroma  and  passes  into  the  watery  solution.  This  process 
is  called  Jicemolysis. 

A  hsBmolysis  may  also  be  brought  about  by  alternately  freezing  and 
thawing  the  blood,  as  well  as  by  the  action  of  various  chemical  subst-'nces, 
which  act  as  protoplasmic  poisons.  These  bodies  are  ether,  chlo.  .orm, 
alkalies,  bile-acids,  solanin,  saponin,  and  also  the  saponin  substances,  which 
have  a  very  strong  hsemolytic  action.  Of  special  interest  in  this  regard 
are  the  haemolysines,  which  act  like  toxines.  These  hsemolysines  may  be 
metabolic  products  of  bacteria  and  may  be  formed  by  higher  plants  and 
by  animals,  such  as  snakes,  toads,  bees,  spiders,  and  others.  Finally, 
the  hsemolysines  or  globulicidal  bodies,  occurrmg  normally  in  blood-sera 
or  produced  in  the  immunization  of  the  blood,  also  belong  here. 

It  seems  that  haemolysis  is  brought  about  in  various  cases  in  different  ways. 
In  the  haemolysis  by  means  of  water  we  are  probably  dealing  with  a  destruction 
or  rupture  of  the  boundary-layer,  while  such  bodies  as  ether,  chloroform,  alkalies, 
bile-acids,  and  saponin  substances,  which  dissolve  lipoids  or  form  combinations 
therewith,  in  this  way  cause  the  passage  of  the  haemoglobin  to  the  outside 
(KoPPE,  Ransom  and  Kobert,  Peskind,  Pascucci).  The  action  of  other 
haemolysines,  such  as  snake- venom  and  tetanotoxine,  seems  to  be  an  action  con- 
nected with  the  lecithin  (Kyes,  Pascucci  ^). 

'  See  Hamburger,  Osmotischer  Druck  und  lonenlehre,  1902;  Koppe,  Pfluger's 
Arch.,  99  and  107;  Albrecht,  Centralbl.  f.  Physiol.,  19;  Pascucci,  Hofmeister's  Beitrage, 
6;   Rywosch,  Centralbl.  f.  Physiol.,  19. 

^  The  work  of  Hamburger,  Hedin,  Eykman,  Koppe,  and  others  on  isotonism,  and 
the  literature  on  this  subject,  may  be  found  .in  Hamburger,  Osmotischer  Druck  und 
lonenlehre,  1902. 

^  Koppe,  1.  c;  Peskind,  Amer.  Journ.  of  Physiol.,  12;  Ransom  and  Kobert,  cited 
by  Pascucci,  Hofmeister's  Beitrage,  0;  Kyes,  Zeitschr.  f.  physiol.  Chem.,  11,  and  Berl. 
klin.  Wochenschr,,  1904. 


194  THE  BLOOD. 

When  the  haemoglobin  is  separated  from  the  so-called  stroma  by  a  suffi- 
ciently strong  dilution  with  water  the  stroma  is  found  in  the  solution  in  a 
swollen  condition.  By  the  action  of  carbon  dioxide,  by  the  careful  addi- 
tion of  acids,  acid  salts,  tincture  of  iodine,  or  certain  other  bodies,  this 
residue,  rich  in  proteins,  condenses,  and  in  many  cases  the  form  of  the 
blood-corpuscles  may  be  again  obtained.  This  residue,  the  so-called 
ghosts  or  stromata  of  the  blood-corpuscles,  can  also  be  directly  colored 
in  dilute  blood  by  methyl  violet  and  in  this  way  detected  (Koppe),  and 
attempts  have  been  made  to  isolate  it  for  chemical  investigation.  In  the 
following  pages  we  mean  by  the  name  stroma  only  that  residue  that  re- 
mains after  the  removal  of  haemoglobin  and  other  bodies  soluble  in  water. 

To  fsolate  the  stromata  from  the  blood-corpuscles,  they  are  washed  first 
by  diluting  the  blood  with  10-20  vols,  of  a  1-2  per  cent  common  salt 
solution  and  then  separating  the  mixture  by  centrifugal  force  or  by 
allowing  it  to  stand  at  a  low  temperature.  This  is  repeated  a  few  times* 
until  the  blood-corpuscles  are  freed  from  serum.  These  purified  blood- 
corpuscles  are,  according  to  Wooldridge,  mixed  with  5-6  vols,  of  water, 
and  then  a  little  ether  is  added  until  complete  solution  is  obtained.  The 
leucocytes  gradually  settle  to  the  bottom,  a  movement  which  may  be 
accelerated  by  centrifugal  force,  and  the  liquid  which  separates  therefrom 
is  very  carefully  treated  with  a  1  per  cent  solution  of  KHSO4  until  it  is 
about  as  dense  as  the  original  blood.  The  separated  stromata  are  collected 
on  a  filter  and  quickly  washed.  Pascucci,^  on  the  contrary,  treats  the 
mass  of  corpuscles  with  15-20  vols,  of  a  -g-  saturated  ammonium-sulphate 
solution,  allows  the  corpuscles  to  settle,  siphons  off  the  fluid,  repeatedly 
centrifuges,  allows  the  residue  to  dry  quickly  (on  porcelain  plates)  at  the 
ordinary  temperature,  and  then  washes  with  water  until  the  blood-pigments 
and  the  other  soluble  bodies  are  dissolved  out. 

Wooldridge  found  as  constituents  of  the  stromata  lecithin,  cholesterin, 
nucleoalbumin,  and  a  globulin  which,  aceordiag  to  Halliburton,  is  prob- 
ably a  nucleoproteid  which  he  calls  cell-glohulin.  No  nuclein  substances 
or  seralbumiri  or  proteoses  could  be  detected  by  Halliburton  and 
Friend.  According  to  Pascucci,  the  stromata  (from  horse-blood)  consists 
of  ^  cholesterin  and  lecithm  (besides  a  little  cerebroside),  and  §  protein 
substances  and  mineral  bodies.  The  nucleated  red  blood-corpuscles  of 
the  bird  contain,  according  to  Plosz  and  Hoppe-Seyler,^  nvjdein  and  a 
protein  which  swells  to  a  slimy  mass  in  a  10  per  cent  common  salt  solution, 
and  which  seems  to  be  closely  related  to  the  hyaline  substance  {hya- 
line substance  of  Rovida,  see  page  141)  occurring  in  the  lymph-cells.  In 
the  mass    extracted   by  alcohol    from   the   blood-corpuscles   of   the   hen, 

'  Hofmeister's  Beitrage,  6. 

MVooldridge,  Arch.  f.  (Anat.  u.)  Physiol.,  1881,  387;  Halliburton  and  Friend, 
Journal  of  Physiol.,  10;  Halliburton,  ibid.,  18;  Plosz,  Hoppe-Seyler's  Med.  chem, 
Untensuch.,  510. 


THE  RED   CORPUSCLES.  l^cp 

AcKERMAXx  1  found  3.93  per  cent  phosphorus  and  17.2  per  cent  nitrogen ^ 
which  on  calculation  gave  42.10  per  cent  nucleic  acid  and  57.82  per  cent 
hist  one.  The  non-nucleated  red  blood-corpuscles  are,  as  a  rule,  ver}^  poor 
in  protein,  but  are  rich  in  haemoglobin;  the  nucleated  corpuscles  are  richer 
in  protein  and  poorer  in  haemoglobin  than  the  non-nucleated. 

A  gelatinous,  fibrin-like  protein  body  may  be  obtained  from  the  red 
blood-corpuscles  under  certain  circumstances.  This  fibrin-like  mass  has^ 
been  observed  on  freezing  and  then  thawing  the  sediment  of  the  blood- 
corpuscles,  or  on  discharging  the  spark  from  a  large  Leyden  jar  through 
the  blood,  or  on  dissohing  the  blood-corpuscles  of  one  kind  of  animal  in 
the  serum  of  another  (Landois,  strorna-fibrin) ;  i.e.,  in  the  so-called  hcem- 
agglutination,  a  clumping  of  the  red  blood-corpuscles  into  clusters  takes 
place.  This  agglutination  can  be  brought  about  by  bodies  similar  to  the 
hiemolysines  and  also  by  serum  constituents  produced  normally  or  by 
immunization.  It  has  not  been  shown  that  a  fibrin  formation  from  the 
stroma  takes  place.  Fibrinogen  has  only  been  detected  in  the  red  cor- 
puscles of  frogs'  blood  (Alex.  Schmidt  and  Semmer^). 

Closely  related  to. the  anatomical  and  chemical  structure  of  the  erythro- 
cytes is  the  question  which  is  important,  for  the  metabolism  m  the  blood,, 
as  to  the  permeability  of  the  er^^throcytes,  that  is,  their  power  of  taking 
up  substances  of  different  kinds.  On  this  subject  we  have  the  researches 
of  Gruns,  Eykmax,  Overton,  Koppe,  and  especially  those  of  Ham- 
burger and  his  collaborators,  and  of  Hedix.^  As  a  result  of  these 
researches,  it  has  been  shown  that  the  blood-corpuscles  are  completely 
impermeable  for  the  ordinary'  varieties  of  sugar,  for  arabite  and  mannite. 
and,  as  it  appears,  also  for  the  cations  Ca"*"^,  Sr++,  Ba"*"*",  Mg+"'".  On  the 
other  hand,  they  are  permeable  for  NH4+  ions,  as  also  for  acids  and  alkalies.^ 
They  are  also  permeable  for  alcohols  (more  readily  the  fewer  Iwdrox}! 
groups  the  molecule  contains),  aldehydes  (with  the  exception  of  paralde- 
hyde), ketones,  ethers,  esters,  urea,  bile  salts,  and  other  compounds.  They 
are  only  slightly  permeable  for  amino-acids.  Towards  the  neutral  potas- 
sium and  sodium  salts,  according  to  Koppe  and  Hamburger,  the  blood- 
corpuscles  are  impermeable  for  the  cations  K+  and  Na+,  and  permeable, 
on  the  contrar}',  for  the  anions  when  an  exchange  of  an  anion,  for  example 
C03=,  in  the  blood-corpuscles  is  possible  -uith  an  anion  in  the  outer  fluid.. 
for  example  with  Cl~,  Br~,  NOa",  etc.  Hober  ^  has  further  shown  that 
the  blood-corpuscles   are  permeable    for  anions    under    the  influence  df 

'  Zeitschr.  f.  physiol.  Chem.,  43. 

'Landois,  Centralbl.  f.   d.  med.  Wissensch.,  1874,   421;    Schmidt,  Pfliiger's  Arch... 
11,  550-559. 

^  In  regard  to  the  hterature,  see  Hamburger,  Osmotischer  Druck-  und  Ionenlehre_ 
*  See  Hober,  Pfliiger's  Arch.,  101  and  102. 
^Pfliiger's  Arch.,  102 


196  THE  BLOOD. 

carbon  dioxide.  Such  an  exchange  of  ions  can  be  especially  observed, 
according  to  Hamburger,  in  the  erythrocytes  suspended  in  NaCl  solution 
and  treated  with  CO2,  when  the  outer  fluid  becomes  alkaline,  due  to  the 
formation  of  Na2C03  by  the  migration  of  Cl~  ions  into  the  corpuscles  and 
an  outward  migration  of  the  COs^  ions.  For  every  one  bivalent  COa^ 
ion  there  must  migrate  inward  two  imivalent  Cl~  ions;  but  as  every  ion 
irrespective  of  whether  it  is  uni-  or  bivalent  has  the  same  osmotic  pressure, 
therefore  the  osmotic  pressure  of  the  blood-corpuscles  must  be  raised,  and 
hence  a  swelling  up  takes  place,  due  to  their  taking  up  water.  The  question 
as  to  how  far  these  observations  can  be  applied  to  the  blood-corpuscles 
in  their  serum,  i.e.,  to  the  blood,  requires  further  proofs.^ 

The  mineral  bodies  of  the  red  corpuscles  will  be  treated  in  connection 
with  their  quantitative  constitution. 

The  constituent  of  the  blood-corpuscles  existmg  in  greatest  quantity  is 
the  red  pigment  hsemoglobin. 

Blood-pigments. 

According  to  Hoppe-Seyler  the  coloring-matter  of  the  red  blood- 
corpuscles  is  not  in  a  free  state,  but  comljined  with  some  other  substance. 
The  crystalline  coloring-matter,  the  hsemoglobin  or  oxyhsemoglobin,  which 
may  be  isolated  from  the  blood,  is  considered,  according  to  Hoppe-Seyler, 
as  a  cleavage  product  of  this  compound,  and  it  acts  in  many  ways  un- 
like the  questionable  compound  itself.  This  compound  is  insoluble  in 
water  and  uncrj^stallizable.  It  strongly  decomposes  hydrogen  peroxide 
without  being  oxidized  itself;  it  shows  a  greater  resistance  to  certain 
chemical  reagents  (as  potassium  ferricyanide)  than  the  free  coloring- 
matter;  and,  lastly,  it  gives  off  its  loosely  combined  oxygen  much  more  easily 
in  vacuum  than  the  free  pigment.  To  distinguish  between  the  cleavage 
products,  the  hsemoglobin  and  the  oxy haemoglobin,  Hoppe-Seyler  calls 
the  compound  of  the  blood-coloring  matter  of  the  venous  blood-corpuscles 
jjhlehin,  and  that  of  the  arterial  arterin.  Other  investigators,  such  as 
H.  U.  KoBERT  and  Bohr,2  the  latter  calling  the  pigment  of  the  blood- 
corpuscles  hcemochrom,  are  of  a  similar  opinion.  Since  the  above-mentioned 
combinations  of  the  blood-coloring  matters  with  other  bodies,  for  example 
(if  they  really  do  exist)  with  lecithin,  have  not  been  closely  studied,  the 
following  statements  will  apply  only  to  the  free  pigment,  the  haemoglobin. 

The  color  of  the  blood  depends  in  part  on  hcemoglohin  and  in  part  on  a 
molecular  combination  of  this  substance  with  oxygen,  the  oxyhcemoglohin. 

'  See  Petry,  Hofmeister  s  Beitrage,  3. 

2  Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  13,  479;  H.  U.  Robert,  Das  Wirbeltier- 
blut  in  mikro-kristallogr.  Hinsicht,  Stuttgart,  1901;  Bohr,  Centrall)!.  {.  Physiol.,  17, 
p.  688. 


BLOOD   PIGMENTS.  197 

We  find  in  blood  after  asphyxiation  almost  exclusively  haemoglobin,  in 
arterial  blood  disproportionately  large  amounts  of  oxyhemoglobin,  and  m 
venous  blood  a  mixture  of  both.  Blood-coloring  matters  are  found  also  in 
striated  as  well  as  in  cert  am  smooth  muscles,  and  lastly  in  solution  in 
different  invertebrates.  The  quantity  of  haemoglobin  in  human  blood  may 
indeed  be  somewhat  variable  under  different  circumstances,  but  amounts 
to  about  14  per  cent  on  an  average,  or  8.5  grams  for  each  kilo  of  the 
weight  of  the  body. 

Hemoglobin  belongs  to  the  group  of  compound  proteids  and  yields  as 
cleavage  products,  besides  very  small  amounts  of  volatile  fatty  acids  and 
other  bodies,  chiefly  a  protein  globin  and  a  coloring-matter,  hcemochromogen 
(about  4  per  cent),  containing  iron,  which  in  the  presence  of  oxygen  is 
easily  oxidized  into  hcematin. 

As  first  shown  by  Schunck  and  ^Iarchlewski,  and  especially  by  the 
work  of  the  latter,  a  close  relationsliip  exists  between  chlorophyll  and  the 
blood-pigment,  because  a  derivative  of  the  first,  phylloporphyrin,  stands 
veiy  close  in  certain  regards  to  a  derivative  of  the  blood-pigment  hsema- 
toporphyrin.  By  the  mvestigations  of  Nexcki  in  conjunction  ^\ith  March- 
LEWSKi  and  Zaleski,^  it  was  shown  that  hsemopyrol  could  be  prepared 
from  the  derivatives  of  both  the  leaf-pigment  and  the  blood-pigments  by 
reduction.  The  fact  that  chlorophyll  and  blood-pigments  are  closely 
related  and  are  constructed  from  the  same  mother-substance  is  of  the 
greatest  biological  importance. 

The  haemoglobin  prepared  from  different  kinds  of  blood  has  not  exactly 
the  same  composition,  which  seems  to  indicate  the  presence  of  different 
haemoglobins.  The  analyses  by  different  investigators  of  the  haemoglobin 
from  the  same  kind  of  blood  do  not  always  agree  wdth  one  another,  which 
probably  depends  upon  the  somewhat  varying  methods  of  preparation. 
The  follo^\ing  analyses  are  given  as  examples  of  the  constitution  of  different 
hnemoglobms : 

Hffimoglobin  from  the  C  H  N  S  Fe  O  P2O5 

Dog 53.85  7.32  16.17  0.390  0.430  21. S4     (Hoppe-Seyler) 

"    54.57  7.22  16.38  0.568  0.336  20.93      (Jaquet) 

Horse 54.87  6.97  17.310.650  0.470  19.73      (Kossel) 

"      51.15  6.76  17.94  0.390  0.335  23.43      (Zinoffsky) 

Ox 54.66  7.25  17.70  0.447  0.400  19.543    ....  (Hufxer) 

Pig 54.17  7.38  16.23  0.660  0.430  21.360    (Otto) 

" 54.717.38  17.43  0.479  0.399  19.602    (Hufner) 

Guinea-pig 54.12  7.36  16.78  0.580  0.480  20.680    (Hoppe-Seyler) 

Squirrel 54.09  7.39  16.09  0.400  0.590  21.440    " 

Goose 54.26  7.10  16.210.540  0.430  20.690  0.770  " 

Hen 52.47  7.19  16.45  0.857  0.335  22.500  0.197  (Jaquet) 

^  Schunck  and  Marchlewski,  Annal.  d.  Chem.  u.  Pharm.,  278,  284,  288,  290;  Nencki, 
Ber.  d.  deutsch.  chem.  Gesellsch.,  29;  Marchlewski  and  Nencki,  Ber.  d.  d.  chem. 
Gesellsch..  34;  Nencki  and  Zaleski,  ibid.;  Marchlewski,  Chem.  Centralbl.,  1902,  I, 
1016;  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  37. 


198  THE  BLOOD. 

The  question  whether  the  amount  of  phosphorus  in  the  haemoglobin 
from  birds  exists  as  a  contamination  or  as  a  constituent  has  not  been 
-decided.  According  to  Ixoko  the  hseraoglobin  from  goose-blood  consists 
of  a  combination  between  nucleic  acid  and  haemoglobin.  In  the  hserao- 
globin from  the  horse  (Zinoffsky),  the  pig,  and  the  ox  (Hufxer)  we  have 
1  atom  of  iron  to  2  atoms  of  sulphur,  while  in  the  haemoglobin  from  the 
dog  (Jaquet)  the  relation  is  1  to  3.  From  the  data  of  the  elementary 
analysis,  as  also  from  the  amount  of  loosel}-  combined  oxygen,  HtiFNER* 
lias  calculated  the  molecular  w^eight  of  dog-haemoglobin  as  14  129  and  the 
formula  CeseH  1025^^1 64FeS30i8i.  According  to  the  more  recent  determina- 
tions of  HiJFXER  and  Jaquet,^  ox-haemoglobin  contains  an  average  of 
0.336  per  cent  iron,  from  which  a  molecular  weight  of  16  669  may  be  cal- 
culated. The  haemoglobin  from  various  kinds  of  blood  not  only  shows  a 
diverse  constitution,  but  also  a  different  solubility  and  crj-stalline  form, 
and  a  varj-ing  quantity  of  water  of  crj'stallization;  hence  we  infer  that 
there  are  several  kinds  of  haemoglobin.  Bohr  is  a  ver}-  zealous  advocate 
of  this  supposition.  He  has  been  able  to  obtain  haemoglobins  from  dog- 
and  horse-blood,  by  fractional  crj'stallization,  which  had  different  powers 
of  combining  with  oxygen  and  contained  different  quantities  of  iron. 
Hoppe-Seyler  had  already  prepared  two  different  forms  of  haemoglobin 
-crj'stals  from  horse-blood,  and  Bohr  concludes  from  all  these  observations 
that  the  ordinary'  haemoglobin  consists  of  a  mixture  of  different  haemo- 
globins. In  opposition  to  this  statement,  Hufxer ^  has  shown  that  only 
one  haemoglobin  exists  in  ox-blood,  and  that  this  is  probably  true  for 
the  blood  of  many  other  animals. 

Oxyhaemoglobin,  which  has  also  been  called  h.ejlatoglgbulix  or 
H-EiLATOCRYSTALLix.  is  a  molecular  combination  of  haemoglobin  and  oxy» 
gen.  For  each  molecule  of  haemoglobin  1  molecule  of  ox\^gen  is  present; 
^nd  the  amount  of  loosely  combined  oxygen  which  is  united  to  1  gram  of 
haemoglobin  (of  the  ox)  has  been  determined  by  Hufxer*  as  1.34  c.c. 
(calculated  at  0°  C.  and  760  mm.  mercur}-). 

According  to  Bohr,  the  facts  are  different.  He  differentiates  between  four 
oxyhsemoglobins,  according  to  the  quantity  of  oxygen  which  they  absorb,  namely, 
a-.  ;?-,  r-.  and  o-oxyhfemoglobin,  all  having  the  same  absorption-spectrum  and  1 

'  Hoppe-Seyler,  Med.  chem.  Untersuch.,  370;  Jaquet,  Zeitschr.  f.  physiol.  Chem., 
14  296;  Kossel.  ibid.,  2,  150;  Zinoffsky,  ibid.,  10;  Hufner,  Beitr.  z.  Physiol.,  Festschr. 
f.  C.  Ludwig  1887;  74-81,  Joum.  f.  prakt.  Chem.  (N.  F.),  22;  Otto,  Zeitschr.  f.  physiol. 
Chem.,  7;    Inoko,  ibid.,  18. 

'  Arch.  f.  (.\nat.  u.)  Physiol,  1894. 

^  Bohr,  ''Sur  les  combinaisons  de  I'hemoglobine  avec  I'oxygene,"  Ext  rait  du 
Bulletin  de  TAcademie  Royale  Danoise  des  sciences,  1890;  also  Centralbl.  f.  Physiol., 
1890,  249.  Hoppe-Seyler,  Zeitschr.  f  physiol.  Chem.,  2;  H  fner.  Arch.  f.  (Anat.  u.) 
Physiol.,  1894. 

*  Arch,  f.  (.\nat   u.)  Physiol..  1901,  Suppl. 


OXYHiEMOGLOBIX.  199 

gram  combining  with  respectively  0.4,  O.S,  1.7,  and  2.7  c.c.  oxygen  at  the  tem- 
perature of  the  room  and  with  an  oxj'gen  pressure  of  150  mm.  mercury.  The 
/-oxyhsemoglobin  is  the  ordinary  one  obtained  by  the  customary  method  of 
preparation.  Bohr  designates  as  a-oxyhsemoglobin  the  crystalline  powder 
obtained  by  drying  /--oxyhsemoglobin  in  the  air.  On  dissolving  a-oxyhaemo- 
gJobin  in  water  it  is  converted  into  ,5-oxyhaemoglobin  witliout  decomposition,  and 
the  quantity  of  iron  is  increased.  On  keeping  a  solution  of  r-oxyhsemoglobin 
in  a  sealed  tube  it  is  transformed  into  o-oxyhsemoglobin,  although  the  exact 
conditions  under  which  this  change  takes  place  are  not  known.  According  to 
HuFNER  '  these  are  nothing  but  mixtures  of  genuine  and  partly  decomposed 
haemoglobins. 

The  ability  of  hsemoglobin  to  take  up  oxygen  seems  to  be  a  function  of 
the  iron  it  contains,  and  when  this  is  calculated  as  about  0.33-0.40  per 
cent,  then  1  atom  of  iron  in  the  hsemoglobin  corresponds  to  about  2  atoms 
or  1  molecule  of  oxygen.  By  increasing  the  partial  pressure  as  well  as 
by  increasing  the  quantities  of  oxygen,  the  hsemoglobin  in  solution  takes 
up  more  oxygen,  until  it  is  completely  saturated,  when  1  molecule  of 
ha?moglobin  is  combined  v\ith  1  molecule  of  oxygen.  Still  this  reaction 
is  reversible  according  to  the  type  1  (Hb)  + 1  (Oo)  ?^  1  (OHb) ,  and  v\ith 
diminished  oxygen  pressure  a  dissociation  must  take  place  with  the  gi^'ing 
up  of  oxygen  and  a  re-formation  of  hsemoglobin.  The  equilibrium  between 
oxyhsemoglobin,  hsemogloljin,  and  oxygen  is  determined  according  to  the 
law  of  mass-action,  and  according  to  the  investigations  of  Hufxer  it  is 
possible  to  calculate  the  relationship  between  ox^-hsemoglobin  (OHb)  and 
hsemoglobin  (Hb),  at  ever}-  desired  partial  pressure  of  the  ox^-gen,  by  a 
formula  suggested  by  him.  According  to  Bohe^  this  formula  does  not 
have  suflBcient  basis  and  does  not  correspond  to  the  facts.  Bohr  found, 
m  opposition  to  Hufxer's  statements,  that  with  the  same  oxygen  tension 
the  absorption  of  oxygen  by  a  hsemoglobin  solution  changes  with  the  con- 
centration, and  that  a  dilute  solution  combines  with  more  oxygen,  calculated 
per  1  gram  hsemoglobin.  than  a  concentrated  solution.  Bohr  suggested 
another  formula  expressing  the  relationship  between  the  ox^-gen  absorp- 
tion and  the  oxygen  tension,  based  upon  the  assumption  that,  besides  the 
dissociation  of  the  oxygen-hsemoglobin  compound,  a  dissociation  of  the 
hsemoglobin  into  a  part  containing  iron  and  a  part  not  containing  iron  also 
takes  place.  This  formula,  which  in  fact  accords  well  with  Bohr's  findings, 
is  nevertheless  only  true  for  a  hsemoglobin  solution  and  not  for  blood,  as, 
according  to  Bohr,  the  blood-pigment  in  the  blood-corpuscles  (the  haemo- 
chrom)  is  changed  on  being  converted  into  hsemoglobin.  Hexri  also 
finds  that  Hufxer's  formula  for  the  dissociation  of  oxyhsemoglobin  is 
not  useful,  basing  his  claim  upon  theoretical  considerations  and  upon 
unfinished  investigations. 

'Arch.  f.   (Anat.  u. )  physiol.,  1894. 

^  Bohr,  Centralbl.  f.  Physiol.,  17  pp.  682  and  688;  Henri,  Compt.  rend.  toe.  biolog., 
o6. 


200  THE  BLOOD. 

The  native  pigment,  the  haemochrom,  combines,  according  to  Bohr,  in 
maximo  with  the  same  quantity  of  oxygen  as  the  corresponding  haemo- 
globin, when  the  latter  is  prepared  without  the  use  of  means  ha\'ing  a  strong 
action;  still  from  this  it  does  not  follow  that  the  oxygen  combination  in 
haemochrom  is  identical  with  that  in  haemoglobin.  According  to  Bohr 
this  is  not  the  case,  at  least  with  diminished  pressure,  for  with  low  oxygen 
tension  more  oxygen  is  taken  up  by  the  blood  than  by  a  corresponding 
haemoglobin  solution.  The  curve  showing  the  oxygen  absorption  is  lower 
in  this  case  for  a  haemoglobin  solution  than  for  blood.  The  reason  for 
this  lies,  according  to  Bohr,  in  the  fact  that  the  tension  curve  is  influenced 
by  the  form  of  union  of  the  part  of  the  haemoglobin  containing  iron  with 
the  iron-free  part,  and  that  this  union  is  changed  because  of  changes  in 
the  iron-free  part,  as  by  the  splitting  off  of  lecithin,  etc.  The  tension 
curve  of  the  oxygen  in  the  blood  can,  according  to  Bohr,  be  determined  only 
by  direct  experiments  on  the  blood  itself  and  not  by  experiments  upon 
haemoglobin  solutions. 

The  elucidation  of  these  conditions  is  of  the  very  greatest  importance, 
as  the  dependence  of  the  reaction  between  OHb,  Hb,  and  O  upon  the  law 
of  mass-action  is  naturally  of  the  very  greatest  moment  for  the  taking 
up  of  oxygen  in  the  lungs  and  the  giving  up  of  the  same  to  the  tissues. 
The  dissociation  of  the  oxy haemoglobin  makes  it  also  possible  to  completely 
expel  the  oxygen  from  a  haemoglobin  solution  or  from  blood  by  means  of 
a  vacuum  or  by  passing  an  indifferent  gas  through  the  blood. 

Oxy  haemoglobin,  which  is  generally  considered  as  a  weak  acid,  is  dextro- 
rotatory, according  to  Gamgee.^  The  specific  rotation  for  light  of  medium 
wave-lengths  of  C  is  (a)C=  about  -1-10°,  which  corresponds  also  for  carbon- 
monoxide  haemoglobin.  The  haemoglobin  is  also,  like  carbon-monoxide 
haemoglobin  (COHb)  and  met  haemoglobin  (MHb),  diamagnetic,  while  the 
haematin,  which  is  richer  in  iron,  is  strongly  magnetic  (Gamgee^).  On 
passing  an  electric  current  through  an  oxyhaemoglobin  solution,  the  pig- 
ment first  separates  unchanged  at  the  anode  in  a  colloidal  but  still  soluble 
form,  and  is  then  gradually  transferred  to  the  cathode  in  the  colloidal 
state  (Gamgee^).  This  transportation  of  the  colloidal  haemoglobin  may 
also  be  made  to  take  place  through  an  animal  membrane  or  through  parch- 
ment paper.  According  to  Gamgee,  the  haemoglobin  probably  exists  in 
such  a  colloidal  condition  in  the  blood-corpuscles. 

Oxyhaemoglobm  has  been  obtained  in  ciystals  from  several  varieties 
of  blood.  These  crj'stals  are  blood-red,  transparent,  silky,  and  may  be 
2-3  mm.  long.  The  oxyhaemoglobin  from  squirrel's  blood  crystallizes 
in  six-sided  plates  of  the  hexagonal  system;  the  other  varieties  of  blood 
yield  needles,  prisms,  tetrahedra,  or  plates  which  belong  to  the  rhombic 

'  Hofmeister's  Beitrage,  4.  '  Proceedings  of  Roy.  Society,  68.  ^  Ibid.,  70. 


OXYH.EMOGLOBIN.  201 

system.i  The  quantity  of  water  of  ctystallization  varies  between  3-10 
per  cent  for  the  different  oxyhaemoglobins.  When  completely  dried  at  a 
low  temperature  over  sulphuric  acid  the  crystals  may  be  heated  to  110-115° 
C.  without  decomposition.  At  higher  temperatures,  somewhat  above 
160°  C,  they  decompose,  giving  an  odor  of  burnt  horn,  and  leave,  after 
complete  combustion,  an  ash  consisting  of  oxide  of  iron.  The  oxyhsemo- 
globm  cr}'stals  from  difficultly  crj'stallizable  kinds  of  blood,  for  example 
from  such  as  ox's,  human,  and  pig's  blood,  are  easily  soluble  m  water^ 
The  oxyhaemoglobins  from  easily  cr}'stallizable  blood,  as  from  that  of  the 
horse,  dog,  squirrel,  and  guinea-pig,  are  soluble  with  difficulty  in  the  order 
above  given.  The  oxyhsemoglobin  dissolves  more  easily  in  a  very  dilute 
solution  of  alkali  carbonate  than  in  pure  water,  and  this  solution  may  be 
kept.  The  presence  of  a  little  too  much  alkali  causes  the  oxy haemoglobin 
to  quickly  decompose.  The  cry^stals  are  insoluble  without  decolorization 
in  absolute  alcohol.  According  to  Nencki,^  it  is  hereby  converted  into 
an  isomeric  or  polymeric  modification,  called  by  him  parahcBmoglohin. 
Oxy  haemoglobin  is  msoluble  in  ether,  chloroform,  benzene,  and  carbon 
disulphide. 

A  solution  of  oxyhaemoglobin  in  water  is  precipitated  by  many  metallic 
salts,  but  is  not  precipitated  by  sugar  of  lead  or  basic  lead  acetate.  On 
heating  the  watery  solution  it  decomposes  at  about  70°  C,  and  splits  off 
protein  and  haematin.  It  is  also  readily  decomposed  by  acids,  alkalies, 
and  many  metallic  salts.  It  gives  the  ordinary  reactions  for  proteins 
with  those  protein  reagents  which  first  decompose  the  oxyhaemoglobin 
with  the  splitting  off  of  protein.  Oxyhaemoglobin,  like  the  other  blood- 
pigments,  has  a  direct  oxidizing  action  upon  tincture  of  guaiacum.  It 
has,  on  the  other  hand,  like  all  blood-pigments  containing  iron,  the  property 
of  an  "ozone  transmitter"  in  that  it  turns  tincture  of  guaiacum  blue  in 
the  presence  of  reagents  containing  peroxide,  such  as  old  turpentine. 

A  sufficiently  dilute  solution  of  oxyhaemoglobin  or  arterial  blood  shows 
a  spectrum  with  two  absorption-bands  between  the  Frauxhofer  lines  D 
and  E.  The  one  band,  a,  which  is  narrower  but  darker  and  sharper,  lies 
on  the  line  D;  the  other,  broader,  less  defined  and  less  dark  band,  /?,  lies 
at  E.  The  middle  of  the  first  band  corresponds  to  a  wave-length  >^  =  578.1 
and  the  second  /^  =  541.7.  These  bands  can  be  detected  in  a  layer  1  cm. 
thick  of  a  0.1  p.  m.  solution  of  oxyhaemoglobin.  In  a  still  weaker  dilution 
the  band  B  first  disappears.     By  increased  concentration  of  the  solution 


'The  observation  of  Uhlik  (Pfliiger's  Arch.,  104)  that  the  haemoglobin  from 
horse-blood  can  also  crystalMze  in  hexagonal  six-sided  plates  seems  to  be  due  to  the 
fact  that  he  had  haemoglobin  and  not  oxyhs&moglobin. 

'  Nencki  and  Sieber,  Ber.  d.  d.  chem.  Gesellsch.,  18.  According  to  Kriiger  (see 
Biochem  Centralbl.,  I,  40,  463)  haemoglobin  is  somewhat  changed  by  alcohol  as  well 
as  by  chloroform. 


202  THE  BLOOD. 

the  two  bands  become  broader,  the  space  between  them  smaller  or  entirely 
obliterated,  and  at  the  same  time  the  blue  and  violet  part  of  the  spectrum 
is  darkened.  The  oxy haemoglobin  may  be  differentiated  from  other  color- 
ing-matters having  a  similar  absorption-spectrum  by  its  behavior  towards 
reducing  substances.^     (See  p.  203.) 

The  observation  of  Piettre  and  Vila  that  so-called  laky  blood  and  oxyhsemo- 
globin  solutions  in  thick  layers  also  show  a  third  band  in  the  red  (A  =  634)  depends 
ill  all  probability,  as  also  claimed  by  Ville  and  Derrien,^  upon  a  partial  forma- 
tion of  methsemoglobin. 

A  great  many  methods  have  been  proposed  for  the  preparation  of 
oxy  haemoglobin  crystals,  but  in  their  chief  features  they  all  agree  with 
the  following  one  suggested  by  Hoppe-Seyler:  The  washed  blood-cor- 
puscles (best  those  from  the  dog  or  the  horse)  are  stirred  with  2  vols. 
water  and  then  shaken  with  ether.  After  decanting  the  ether  and  allowing 
the  ether  which  is  retained  by  the  blood  solution  to  evaporate  in  an  open 
dish  in  the  air,  cool  the  filtered  blood  solution  to  0°  C,  add  while  stirring 
J  vol.  of  alcohol  also  cooled,  and  allow  to  stand  a  few  days  at  —5°  to  — 10° 
C.  The  crj^stals  which  separate  may  be  repeatedly  recrystallized  by 
dissolving  in  water  of  about  35°  C,  cooling,  and  adding  cooled  alcohol  as 
above.  Lastly,  they  are  washed  with  cooled  water  containing  alcohol 
(I  vol.  alcohol)  and  dried  in  vacuum  at  0°  C.  or  a  lower  temperature.-^ 

For  the  preparation  of  oxyhsemoglobin  crystals  in  small  quantities 
from  easily  crystallizable  blood,  it  is  often  sufficient  to  stir  a  drop  of  blood 
with  a  little  water  on  a  microscope  slide  and  allow  the  mixture  to  evaporate 
so  that  the  drop  is  surrounded  by  a  dried  ring.  After  covering  with  a 
cover-glass,  the  crystals  gradually  appear  radiating  from  the  ring.  These 
crj'stals  are  formed  more  surely  if  the  blood  is  first  mixed  with  some  water 
in  a  test-tube  and  shaken  with  ether  and  a  drop  of  the  lower  deep-colored 
liquid  treated  as  above  on  the  slide. 

Haemoglobin,  also  called  reduced  hemoglobin  or  purple  cruorin 
(Stores'*),  occurs  only  in  very  small  quantities  in  arterial  blood,  in  larger 
quantities  in  venous  blood,  and  is  nearly  the  only  blood-coloring  matter 
after  asphyxiation. 

Haemoglobin  is  much  more  soluble  than  the  oxy  haemoglobin,  and  it  can 
therefore  be  obtained  as  crystals  only  with  difficulty.  These  crystals  are 
as  a  rule  isomorphous  with  the  corresponding  oxyhaemoglobin  crystals, 
but  are  darker,  having  a  shade  towards  blue  or  purple,  and  are  decidedly 

'  Zeitschr.  f.  Biologie,  34,  contains  the  investigations  of  Gamgee  on  the  absorp- 
tion of  the  ultra-violet  rays  by  the  blood  pigment.  It  also  contains  some  of  the  earlier 
investigations. 

^  Piettre  and  Vila,  Compt.  rend.,  140;   Ville  and  Derrien,  ibid.,  140. 

'  In  regard  to  the  preparation  of  oxyhsemoglobin,  see  also  Hoppe-Seyler-Thier- 
felder's  Handbuch,  7.  Aufl.;  also  the  works  cited  in  foot-note  1,  p.  198;  also  Schuur- 
manns-Stekhoven,  Zeitschr.  f.  physiol.  Chem.,  33,  296;  see  also  Bohr,  Skand.  Arch, 
f.  Physiol.,  3. 

*  Philosophical  Magazine,  28,  No.  190,  Nov.,  1864. 


HEMOGLOBIN.  203 

more  pleochromatic.  The  hsemoglobin  from  horse-blood  has  also  been 
obtained  by  Uhlik  ^  in  hexagonal  six-sided  plates.  Its  solutions  in  water 
are  darker  and  more  \dolet  or  purplish  than  solutions  of  oxyhsemoglobin 
of  the  same  concentration.  They  absorb  the  blue  and  the  violet  rays  of 
the  spectrum  in  a  less  marked  degree,  but  strongly  absorb  the  rays  lying 
between  C  and  D.  In  proper  dilution  the  solution  shows  a  spectrum  with 
one  broad,  not  sharply  defined  band  between  D  and  E,  whose  darkest  part 
corresponds  to  the  wave-length  ^  =  555.  Tliis  band  does  not  lie  in  the 
middle  between  D  and  E,  but  is  towards  the  red  end  of  the  spectrum,  a 
little  over  the  line  D.  A  haemoglobin  solution  actively  absorbs  oxygen 
from  the  air  -^ni  is  converted  into  an  oxyhsemoglobin  solution. 

A  solution  of  oxyhsemoglobin  may  be  easily  converted  into  a  solution 
having  the  spectrum  of  hsemoglobin  by  means  of  a  vacuum,  by  passing  an 
indifferent  gas  through  it,  or  by  the  addition  of  a  reducing  substance,  as, 
for  example,  an  ammoniacal  ferrous-tartrate  solution  (Stokes'  reduction 
liquid).  If  an  oxyha?moglobin  solution  or  arterial  blood  is  kept  in  a  sealed 
tube,  we  observe  a  gradual  consumption  of  oxygen  and  a  reduction  of  the 
oxyhsemoglobin  into  hsemoglobin.  If  the  solution  has  a  proper  concen- 
tration, a  crj'stallization  of  hsemoglobin  may  occur  in  the  tube  at  lower 
temperatures  (Hufner^). 

Pseudohaemoglobin.  Ludwig  and  Siegfried  '  have  observed  that  blood 
which  has  been  reduced  by  hyposulphites  so  completely  that  the  oxyhsemoglobin 
spectrum  disappears  and  only  the  haemoglobin  spectrum  is  seen,  yields  large 
amounts  of  oxygen  when  exposed  to  a  vacuum.  Blood  which  has  been  reduced 
by  the  passage  of  a  stream  of  hydrogen  through  it  until  the  oxyhsemoglobin 
spectrum  disappears  acts  in  the  same  manner.  Hence  a  loose  combination  of 
hsemoglobin  and  oxygen  exists  which  gives  the  hsemoglobin  spectrum,  and  this 
combination  is  called  pseudohsemoglobin  by  Ludwig  and  Siegfried.  Pseudo- 
haemoglobin,  whose  presence  has  been  detected  in  asphyxiation  blood  from  dogs, 
is  considered  by  Hammarsten  as  an  intermediate  step  between  hsemoglobin  and 
oxyhsemoglobin  on  the  reduction  of  the  latter.  The  occurrence  of  pseudohsemo- 
globin does  not  seem  to  have  been  positively  proved.* 

Methaemoglobin.  This  name  has  been  given  to  a  coloring-matter  which 
is  easily  obtained  from  oxyhsemoglobin  as  a  transformation  product  and 
which  has  been  correspondingly  found  in  transudates  and  cystic  fluids 
containing  blood,  in  urine  in  haematuria  or  hsemoglobinuria,  also  in  urine 
and  blood  on  poisoning  with  potassium  chlorate,  amyl  nitrite  or  alkali 
nitrite,  and  many  other  bodies. 

Methaemoglobin  does  not  contain  any  oxygen  in  molecular  or  dissociable 
combination,  but  still  the  oxygen  seems  to  be  of  importance  in  the  forma- 
tion of  methsemoglobin,  because  it  is  formed  from  0x3- hsemoglobin  and 
not  from  hsemoglobin  in  the  absence  of  oxygen  or  oxidizing  agents.    If 

» Pfliiger's  Arch.,  104. 

^  Zeitschr.  f.  physiol.  Chem.,  4;   see  also  Uhlik,  1.  c. 

^  Arch.  f.  (Anat.  u.)  Physiol.,  1890;  see  also  Ivo  Novi,  Pfliiger's  Archiv,  56. 

*  See  Hufner,  Arch,  f .  (Anat.  u.)  Physiol.,  1894, 140. 


204  THE  BLOOD. 

arterial  blood  be  sealed  up  in  a  tube,  it  gradually  consumes  its  oxj^gen  and 
becomes  venous,  and  by  tliis  absorption  of  oxygen  a  little  methsemogiobin 
is  formed.  The  same  occurs  on  the  addition  of  a  small  quantity  of  acid  to 
the  blood.  By  the  spontaneous  decomposition  of  blood  some  methsemo- 
giobin is  formed,  and  by  the  action  of  ozone,  potassium  permanganate, 
potassium  ferricyanide,  chlorates,  nitrites,  nitrobenzene,  pyrogallol,  pyro- 
cateehin,  acetanilide,  and  certain  other  bodies  on  the  blood  an  abundant 
formation  of  methamoglobin  takes  place. 

According  to  the  investigations  of  Hufner,  Kulz,  and  Otto  ^  methsemo- 
giobin contains  just  as  much  oxygen  as  oxyhsemoglobm,  but  it  is  more 
strongly  coml3ined.  By  the  action  of  potassium  ferricyanide  or  potassium 
permanganate  upon  oxy haemoglobin  first  1  molecule  oxygen  (i.e.,  the 
entire  quantity  of  loosely  combined  oxygen)  is  split  off  and  in  the  subse- 
quent methsemogiobin  formation  either  two  oxygen  atoms  (Haldane)  or 
two  hydroxyl  groups  are  combined  (Hufner,  v.  Zeyxek^).  ^ilethsemo- 
globin  solutions  are  reduced  to  haemoglobin  by  reducing  agents.  Jader- 
HOLM  and  Saarbach  claim  that  methsemogiobin  is  first  converted  into 
oxyhsemoglobin  and  then  into  hsemoglobin  by  reducing  substances,  while 
others  (Hoppe-Seyler  and  Araki^)  dispute  this. 

According  to  HiJFNER  and  Reinbold*  1  gram  methsemogiobin  can 
take  up  2.685  c.c.  nitric  oxide. 

^lethsemoglobin  cr}^stallizes,  as  first  showTi  by  HiJFXER  and  Otto,  in 
bro\\Tiish-red  needles,  prisms,  or  six-sided  plates.  It  dissolves  easily  in 
water;  the  solution  has  a  brown  color  and  becomes  a  beautiful  red  on  the 
addition  of  alkali.  The  solution  of  the  pure  substance  is  not  precipitated 
by  basic  lead  acetate  alone,  but  by  basic  lead  acetate  and  ammonia.  The 
absorption-spectrum  of  a  watery  or  acidified  solution  of  methsemogiobin  is, 
according  to  Jaderholm  and  Bertin-Saxs,  ver\'  similar  to  that  of  hsematin 
in  acid  solution,  but  is  easily  distmgiiished  from  the  latter  since,  on  the 
addition  of  a  little  alkali  and  a  reducing  substance,  the  former  jDasses 
over  to  the  spectnun  of  reduced  hsemoglobin,  while  a  hsematin  solution 
under  the  same  conditions  gives  the  spectrum  of  an  alkaline  hsemochromogen 
solution  (see  below).  [Methsemogiobin  in  alkaline  solution  shows  two 
absorption-bands  which  are  like  the  two  oxyhsemoglobin  bands,  but  they 
differ  from  these  in  that  the  band  /?  is  stronger  than  a.  By  the  side  of 
the  band  a  and  united  with  it  by  a  shadow  lies  a  third  fainter  band  between 
C  and  D,  near  to  D.     According  to  other  investigators.  Araki  and  Dit- 

*  See  Otto,  Zeitschr.  f.  physiol.  Chem.,  7. 

2  Haldane,  Journ.  of  Physiol.,  22;  v.  Zeynek,  Arch.  f.  (Anat.  u.)  Physiol.,  1899; 
Hiifner,  ibid. 

^  Jaderholm,  Zeitschr.  f.  Biologie,  16;  Saarbach,  Pfliiger'.'^  Arch.,  28;  Araki,  Zeit- 
schr. f.  phy.siol.  Chem.,  14. 

*  Arch.  f.  (Anat.  u.)  Physiol.,  1904,  Suppl. 


CARBOX-xAIOXOXIDE  HiE:\IOGLOBIN.  205 

TRICH,  a  neutral  or  faintly  acid  methsemoglobin  solution  shows  only  one 
characteristic  band,  a,  between  C  and  D,  whose  middle  corresponds  to 
about  i^  =  634.  The  two  bands  between  D  and  E  are  only  due  to  con- 
tamination with  oxyhsemoglobin  (Menzies^). 

The  statements  as  to  the  action  of  sodium  fluoride  \\y>o\\  haemoglobin  and 
naethaemoglobin  are  somewhat  contradictory. - 

Crv'stallized  methoemoglobin  may  be  easily  obtained  by  treating  a  con- 
centrated solution  of  oxyha^moglobin  with  a  sufficient  c^uantity  of  concen- 
trated potassium-ferrieyanide  solution  to  give  the  mixture  a  porter-brown 
color.  After  cooling  to  0°  C.  add  \  vol.  cooled  alcohol  and  allow  the  mix- 
ture to  stand  a  few  days  in  the  cold.  The  ciystals  may  be  easily  purified 
by  recrv'stallizing  from  water  by  the  addition  of  alcohol. 

Cyanmethaemoglobin  (cyanhsemoglobin)  is,  according  to  Haldaxe,  identical 
with  photomethsemoglobin  (Bock),  which  is  produced  by  the  influence  of  sunlight 
upon  a  methsemoglobin  solution  containing  potassium  ferricyanide.  It  was 
first  carefully  described  by  R.  Kobert  and  obtained  in  a  crystalline  form  by 
V.  Zeyxek.^  It  is  immediately  formed  in  the  cold  by  the  action  of  a  hydrocyanic- 
acid  solution  upon  methsemoglobin,  but  is  formed  by  its  action  upon  oxyhsemo- 
globin only  at  the  body  temperature.  The  neutral  or  faintly  alkaline  solutions 
show  a  spectrum  which  is  very  similar  to  the  hsemoglobin  spectrum. 

Acid  haemoglobin  is  a  coloring-matter  produced  by  the  action  of  very  weak 
acids  upon  oxyhsemoglobin,  which  according  to  Harxack  ■*  is  not,  as  used  to  be 
admitted,  identical  with  methsemoglobin. 

Carbon-monoxide  Haemoglobin  ^  is  the  molecular  combination  between 
1  molecule  of  hsemoglobm  and  1  molecule  of  CO,  according  to  Hufner,® 
which  contains  1.34  c.c.  of  carbon  monoxide  (at  0°  and  760  mm.  Hg) 
for  1  gram  hsemoglobin.  Tliis  combmation  is  stronger  than  the  oxygen 
combination  of  hsemoglobin.  The  oxygen  is  for  this  reason  easily  driven 
out  of  oxyhsemoglobin  by  carbon  monoxide,  and  tliis  explains  the  jDoison- 
ous  action  of  tliis  gas,  which  kills  by  the  expulsion  of  the  oxygen  of  the 
blood.  In  regard  to  the  division  of  the  blood-pigments  between  the  carbon 
monoxide  and  oxygen  under  different  partial  pressures  of  both  gases  in 

'  Jaderholm,  1.  c;  Bertin-Sans,  Comp.  rend.,  106;  Dittrich,  Arch.  f.  exp.  Path.  u. 
Pharm.,  29;  Menzies,  Journ.  of  Physiol.,  17.  Important  references  on  methsemo- 
globin are  given  by  Otto,  Pfliiger's  Arch.,  31. 

-  Piettre  and  Vila,  Conipt.  rend.,  140;  Ville  and  Derrien,  ibid..  140. 

^  Haldane,  Journ.  of  Physiol.,  25;  Bock,  Skand.  Arch.  f.  Physiol.,  6;  Kobert, 
Pfliiger's  Arch.,  82;   v.  Zeynek,  Zeitschr.  f.  physiol.  Cheni.,  33. 

■*  Zeitschr.  f.  physiol.  Chem.,  26. 

'In  reference  to  carbon-monoxide  haemoglobin,  see  especially  Hoppe-Seyler,  Med.- 
chem.  Untersuch.,  201;  Centralbl.  f.  d.  med.  Wissensch.,  1S64  and  1865;  Zeitschr. 
f.  physiol.  Chem.,  1  and  13. 

'Arch.  f.  (Anat.  u.)  Physiol.,  1894.  On  the  dissociation  constant  of  carbon- 
monoxide  hsemoglobin,  see  ibid.,  1895.  In  regard  to  the  contradictory  statements  of 
Saint-Martin  and  others  and  their  disproval,  see  Hi  fner,  Arch.  f.  (Anat.  u.)  Physiol., 
1903. 


206  THE  BLOOD. 

the  air,  we  must  refer  to  the  mvestigations  of  HiJFNER,i  whose  results  are 
tabulated. 

The  carbon  monoxide  can  be  driven  out  by  a  vacuum  as  well  as  by 
passing  an  indifferent  gas  or  oxygen  or  nitric  oxide  through  the  solution 
for  a  long  time,  and  in  these  cases  haemoglobin,  oxyhemoglobin,  or  nitric- 
oxide  hsemoglol^in  are  formed.  The  carbon  monoxide  is  also  expelled  by 
potassium  ferricyanide  and  methsemoglobin  is  formed  (Haldane^). 

Carbon-monoxide  haemoglobin  is  formed  by  saturating  blood  or  a 
haemoglobin  solution  with  carbon  monoxide,  and  may  be  obtained  as  crystals 
by  the  same  means  as  oxyhaemoglobin.  These  crystals  are  isomorphous 
with  the  oxyhaemoglobin  cr}'stals,  but  are  less  soluble  and  more  stable, 
and  their  bluish-red  color  is  more  marked.  For  the  detection  of  carbon- 
monoxide  haemoglobin,  its  absorption-spectrum  is  of  the  greatest  importance. 
This  spectnmi  shows  two  bands  which  are  verj'  similar  to  those  of  ox3^haBmo- 
globin,  but  they  occur  more  towards  the  violet  part  of  the  spectrum.  The 
middle  of  the  first  band  corresponds  to  X  =  ^12  and  the  second  to  ^  =  536. 
These  bands  do  not  change  noticeably  on  the  addition  of  reducing  sub- 
stances; this  constitutes  an  important  difference  between  carbon-monoxide 
haemoglobin  and  oxyhaemoglobin.  If  the  blood  contains  oxyhaemoglobin. 
and  carbon-monoxide  haemoglobin  at  the  same  time,  we  obtain  on  the 
addition  of  a  reducing  substance  (ammoniacal  ferro-tartrate  solution) 
a  mixed  spectrum  originating  from  the  haemoglobin  and  carbon-monoxide 
haemoglobin. 

A  great  many  reactions  have  been  suggested  for  the  detection  of  carbon- 
monoxide  haemoglobin  in  medico-legal  cases.  A  simple  and  at  the  same 
time  a  good  one  is  Hoppe-Seyler's  alkali  test.  The  blood  is  treated  with 
double  its  volume  of  caustic-soda  solution  of  1.3  sp.  gr.,  by  which  ordinary 
blood  is  converted  into  a  dingy  brownish  mass,  which  when  spread  out 
on  porcelain  iS  brown  with  a  shade  of  green.  Carbon -monoxide  blood 
gives  under  the  same  conditions  a  red  mass,  which  if  spread  out  on  porce 
lain  shows  a  beautiful  red  color.  Several  modifications  of  this  test  have 
been  proposed.  Another  very  good  reagent  is  tannic  acid,  which  gives 
with  dilute  normal  blood  a  brownish-green  precipitate  and  with  carbon- 
monoxide  blood  a  pale  crimson-red  precipitate.^ 

As  according  to  Bohr  there  are  several  oxj'^hsemoglobins,  so  also,  according  to 
Bohr  and  Bock,''  there  are  several  carbon-monoxide  haemoglobins,  with  different 


'  Arch.  f.  exp.  Path.  u.  Pharm.,  48. 

'  Journ.  of  Physiol.,  22. 

'  In  regard  to  this  test  (as  suggested  by  Kunkel)  and  others  we  refer  to  Kostin, 
Pfii!ger's  Arch.,  S-i,  which  contains  a  very  excellent  summary  of  the  literature  on  the 
subject. 

*  Centralbl.  f.  Physiol.,  8,  and  Maly's  Jahresber.,  25. 


CARBON-DIOXIDE  HAEMOGLOBIN.  207 

amounts  of  carbon  monoxide.  As  heemoglobin  can  unite  with  oxygen  and  carbon 
dioxide  simultaneously,  as  shown  by  Bohr  and  Torup,  so  also  can  it  unite  with 
carbon  monoxide  and  carbon  dioxide  simultaneously  and  independently  of  each 
other. 

Carbon-monoxide  methaemoglobin  has  been  prepared  by  Weil  and  v.  Anrep 
by  the  action  of  potassium  permanganate  on  carbon-monoxide  haemoglobin,, 
but  this  is  contradicted  by  Bertin-Sans  and  Moitessier.'  Sulphur  methaemo- 
globin is  the  name  given  by  Hoppe-Seyler  to  that  colormg-matter  which  is 
formed  by  the  action  of  sulphuretted  hydrogen  upon  oxyhaemoglobin.  The 
solution  has  a  greenish-red,  dirty  color,  and  shows  two  absorption-bands  between 
C  and  D.  This  coloring-matter  is  claimed  to  be  the  greenish  color  seen  on  the 
surface  of  putrefying  flesh.  According  to  Harnack  ^  the  conditions  are  different 
when  HjS  is  passed  through  an  oxygen-free  solution  of  haemoglobin  (or  carbon- 
monoxide  haemoglobin).  The  sulphhaemoglobin  thus  formed  shows  one  band  ia 
the  red  between  C  and  D. 

Carbon-dioxide  Haemoglobin,  Carbohceinoglohin.  Hsemoglobin,  accord- 
ing to  Bohr  and  Torup,^  also  forms  a  molecular  combination  with  carbon 
dioxide  whose  spectrum  is  similar  to  that  of  hemoglobin.  According  ta 
Bohr  there  are  three  different  carbohsemoglobins,  namely,  a-,  /9-,  and 
;--carboha3moglobin,  in  which  1  gram  combines  with  respectively  1.5,  3,  and 
6  c.c.  CO2  (measured  at  0°  C.  and  760  mm.)  at  18°  C.  and  a  pressure  of  60 
mm.  mercury.  If  a  hsemoglobin  solution  is  shaken  with  a  mixture  of 
oxygen  and  carbon  dioxide,  the  hsemoglobin  combines  loosely  with  the 
oxygen  as  well  as  with  the  carbon  dioxide,  independently  of  each  other,. 
just  as  if  each  gas  existed  alone  (Bohr).  He  considers  that  the  two  gases 
are  combined  with  different  parts  of  the  hsemoglobin,  that  is,  the  oxygen 
with  the  pigment  nucleus  and  the  carbon  dioxide  with  the  protein  com- 
ponent. Bohr  has  given  an  equilibrium  formula  for  the  carbon-dioxide 
absorption  of  hsemoglobin  at  different  carbon-dioxide  tensions,  and  the 
results  obtained  on  calculation,  using  this  formula,  correspond  veiy  well 
with  the  results  obtained  directly.  Attention  must  be  called  to  the  fact 
that,  as  observed  by  Torup,  hsemoglobin  is  in  part  readily  decomposed 
by  the  carbon  dioxide  with  the  splitting  off  of  some  protein. 

Nitric-oxide  Haemoglobin  is  also  a  crv'stalline  molecular  combination 
which  is  even  stronger  than  the  carbon-monoxide  hsemoglobin.  Its  solu- 
tion shows  two  absorption-bands  which  are  paler  and  less  sharp  than  the 
carbon-monoxide  hsemoglobin  bands,  and  they  do  not  disappear  on  the 
addition  of  reducing  bodies.  Hsemoglobin  also  forms  a  molecular  com- 
biration  wHh  acetylene. 

Hasmorrhodin  is  the  name  given  by  Lehmann  to  a  beautiful  red  pigment 
soluble  in   alcohol  and  ether,  which  is  extracted  from  meat  and  meat  products 

^  V.  Anrep,  Arch.  f.  (Anat.  u.)  physiol.,  1880;  Sans  and  Moitessier,  Compt.  rend.,  113. 

^  Med.-chem.  Untersuch.,  151.  See  Araki,  Zeitschr.  f.  physiol.  Chem.,  11;  Har- 
nack, 1.  c. 

5  Bohr,  Extrait  du  Bull,  de  I'Acad.  Danoise,  1890;  Centralbl.  f.  Physiol.,  4  ami 
17;    Torup,  Maly's  Jahresber.,  17. 


208  THE  BLOOD. 

by  boiling  alcohol  and  which  seems  to  be  produced  by  the  action  of  small  amounts 
of  nitrites.  Another  pigment  isolated  by  Lewin  '  from  the  blood  of  animals 
poisoned  by  phenylhydrazine  has  been  called  hcemoverdin.  By  heating  a  solu- 
tion of  blood-pigment  treated  with  caustic  potash  and  mixed  with  alcohol  to 
60°  C.  we  obtain,  according  to  v.  Klaveren,  a  pigment  which  he  calls  kathcemo- 
globin,  but  called  by  Arnold,^  who  first  obtained  it,  neutral  hcematin,  which  is 
produced  by  the  splitting  off  of  a  ferruginous  complex.  This  pigment  still  con- 
tains protein,  but  is  poorer  in  iron  than  the  hsemoglobin  or  methsemoglobin  and 
probably  forms  an  intermediary  product  in  the  conversion  of  the  above  into 
haematin. 

Decomposition  j/rodiicts  of  the  blood-pigtnents.  By  its  decomposition 
haemoglobin  yields,  as  pre^^ollsly  stated,  a  protein,  which  has  been  called 
globin  (Preyer,  Schulz),  and  a  ferruginous  pigment  as  chief  products. 
According  to  Lawrow  94.09  per  cent  protein,  4.47  per  cent  haematin,  and 
1.44  per  cent  other  bodies  are  produced  in  this  decomposition.  The  globin, 
which  was  isolated  and  studied  by  Schulz,^  differs  from  most  other  pro- 
teins by  containing  a  high  amount  of  carbon,  54.97  per  cent,  with  1698. 
per  cent  of  nitrogen.  It  is  insoluble  in  water,  but  very  easily  soluble  in 
acids  or  alkalies.  It  is  not  dissolved  by  ammonia  in  the  presence  of 
ammonium  chloride.  Nitric  acid  precipitates  it  in  the  cold  but  not  when 
warm.  It  may  be  coagidated  by  heat,  but  the  coagulum  is  readily  soluble 
in  acids.     Because  of  these  reactions  it  is  considered  as  a  histone  by  Schulz. 

On  hydrolytic  cleavage  globin  (from  horse-blood)  yields,  according  to 
Abderhalden,^  the  ordinary  cleavage  products  of  the  proteins  and 
especially  leucine,  29  per  cent.  It  is  also  important  to  call  attention  to.  the 
large  amount  of  histidine,  10.96  per  cent,  while  the  quantities  of  arginine 
and  lysine  were  only  5.42  and  4.28  per  cent  respectively. 

The  pigment  split  off  is  different,  depending  upon  the  conditions  under 
which  the  cleavage  takes  place.  If  the  decomposition  takes  place  in  the 
absence  of  oxygen,  a  coloring-matter  is  obtained  which  is  called  by 
Hoppe-Seyler  h(Bmochromogen,  by  other  investigators  (Stokes)  reduced 
hcematin.  In  the  presence  of  oxygen,  haemochromogen  is  quickly  oxidized 
to  haematin,  and  there  is  therefore  obtained  in  this  case  hcematin  as  a  colored 
decomposition  product.  As  haemochromogen  is  easily  converted  by 
oxygen  into  haematin,  so  this  latter  may  be  reconverted  into  haemochromogen 
by  reducing  substances. 

Haemochromogen  was  discovered  by  Hoppe-Seyler.^  It  is,  accord- 
ing to  Hoppe-Seyler,  the  colored  atomic  group  of  haemoglobin  and  of  its 
combinations  with  gases,  and  this  atomic  group  is  combined  with  proteins 

^  K.  B.  Lehmann,  Sitzungsber.  d.  phys.-med.  Gesellsch.  Wiirzburg,  1899;  Lewin, 
Compt.  rend.,  133. 

^  V.  Klaveren,  Zeitschr.  f.  physiol.  Chem.,  33;  Arnold,  ibid.,  29. 

^Lawrow,  ibid.,  26;   Schulz,  ibid.,  24;  Preyer,  Die  Blutkristalle,  Jena,  1871. 

^  Zeitschr.  f.  physiol.  Chem.,  37. 

'  Ibid.,  13. 


H.EMOCHROMOGEX.  209 

in  the  pigment.  The  characteristic  absorption  of  light  depends  on  tlie 
haemochromogen,  and  it  is  also  tliis  atomic  group  which  binds  in  the  oxy- 
hsemoglobin  1  molecule  of  oxygen  and  in  the  carbon-monoxide  hsemoglobm 
1  molecule  of  carbon  monoxide  ^^■ith  1  atom  of  iron.  Htemochromogen 
is  produced  in  an  alkaline  solution  of  hipmatin  by  the  action  of  reducing 
bodies.  By  the  reduction  of  hsematin  in  alcoholic  ammoniacal  solution  by 
means  of  hydrazine  v.  Zeyxek  ^  was  able  to  obtain  the  solid  bro\\Tiish-red 
ammonia  combination. 

Haemochromogen  combines,  as  Hoppe-Seyler  first  showed,  also  with 
carbon  monoxide.  This  compound,  which  hi  aqueous  solution  gives 
a  spectrum  similar  to  oxy haemoglobin,  has  been  obtained  by  Pregl-  in 
the  solid  condition  as  a  deep-^dolet  powder  which  is  insoluble  in  absolute 
alcohol.  In  opposition  to  haemoglobin  the  haemochromogen  combines 
with  oxygen  more  firmly  than  with  carbon  monoxide.  The  assumption 
of  Hoppe-Seyler  that  this  compound  is  a  combination  of  1  molecule 
haemochromogen  and  therefore  contains  1  molecule  carbon  monoxide 
for  1  molecule  of  iron  has  been  experimentally  substantiated  by  Hufner 
and  KuSTER  and  by  Pregl.3 

An  alkaline  hsmochromogen  solution  has  a  beautiful  cherr\'-red  color. 
It  shows  two  absorption-bands,  first  described  by  Stokes,  one  of  which 
is  dark  and  whose  center  corresponds  to  X  =  oo6A  between  D  and  E, 
and  a  second  broader  band,  less  dark,  which  covers  the  Frauxhofer 
lines  E  and  b.  The  middle  of  this  band  corresponds  to  /  =  520.4.  In  acid 
solution  haemochromogen  shows  four  bands,  which,  according  to  Jader- 
HOLM,*  depend  on  a  mixture  of  haemochromogen  and  haematoporphyrin 
(see  below),  this  last  formed  by  a  partial  decomposition  resulting  from 
the  action  of  the  acid. 

MiLROY  °  from  an  alcoholic  solution  of  hsematm  containing  oxalic  acid, 
after  drivmg  out  the  air  by  means  of  hydrogen  gas,  gradualh'  obtained 
an  acid  solution  of  reduced  haematin  (haemochromogen)  bj'  means  of  zinc 
dust.     This  solution  showed  one  absorption-band  between  D  and  E. 

Haemochromogen  may  be  obtamed  as  cr\-stals  by  the  action  of  caustic 
soda  on  hsemoglobm  at  100°  C.  m  the  absence  of  oxygen  (Hoppe-Seyler). 
By  the  decomposition  of  haemoglobin  by  acids  (of  coui-se  m  the  absence  of 
air)  we  obtain  haemochromogen  contaminated  with  a  little  haematopor- 
phyrin. An  alkaline  haemochromogen  solution  is  easily  obtained  by  the 
action  of  a  reducing  substance  (Stokes'  reduction  liquid)  on  an  alkaline 
haematin  solution.  An  ammoniacal  solution  of  haematin  on  reduction  with 
hydrazine    yields    haemochromogen    veiy    easih'.     An    alcoholic,   alkaline 

'  Zeitschr.  f.  physiol.,  Chem.,  25. 

'Ibid.,U. 

•  H  fner  and  K'  ster.  Arch.  f.  (Anat.  u.)  Physiol.,  1904,  Suppl.;  Pregl,  1.  c. 

*Nord.  Med.  Arkiv.,  16. 

'  Journ.  of  Physiol.,  32. 


210  THE  BLOOD. 

hydrazine  solution  is  also  recommended  by  Riegler  ^  as  a  reagent  for 
blood-pigments,  converting  them  into  hsemochromogen. 

Haematin,  also  called  Oxyh^m-A-Tin,  is  sometimes  found  in  old  transu- 
dates. It  is  formed  by  the  action  of  the  gastric  or  pancreatic  juices  on 
oxy haemoglobin,  and  is  therefore  also  found  in  the  faeces  after  hemorrhage 
in  the  intestinal  canal,  and  also  after  a  meat  diet  and  food  rich  in  blood. 
It  is  stated  that  haematin  may  occur  in  urine  after  poisoning  uith  arseniu- 
retted  hydrogen  As  sho\^Ti  above,  the  haematin  is  formed  by  the  decom- 
position of  oxy  haemoglobin,  or  at  least  of  haemoglobin,  in  the  presence  of 
oxygen. 

The  statements  in  regard  to  the  composition  of  haematin  are  rather 
contradictory,  which  seems  to  be  due  to  the  fact  that  the  substance, 
haemin  (see  below),  from  which  the  formula  of  haematin  is  derived,  has 
a  somewhat  different  composition,  dependent  upon  various  conditions. 
According  to  Hoppe-Seyler  haematin  has  the  formula  C34H34N4Fe05. 
and  from  the  recent  investigations  upon  haemin,  which  will  be  mentioned 
below,  this  formula  seems  to  be  now  generally  accepted.  According  to 
this  formula  1  atom  of  iron  occurs  with  every  4  atoms  of  nitrogen.  Ac- 
cording to  Cloetta,  and  also  RoSENFELDr  haematm  has  the  formula 
C3oH34N3Fe03,  with  1  atom  of  iron  for  ever}'  3  atoms  of  nitrogen. 

Haematin  is  very  resistant  towards  boiling  concentrated  caustic  potash 
as  well  as  towards  boiling  hydrochloric  acid.  It  dissolves  in  concentrated 
sulphuric  acid  and  is  converted  into  haematoporphyrin  with  the  splitting 
off  of  iron.  On  heating  dr}^  haematin  it  yields  abundant  pyrrol.  On 
reduction  with  tin  and  hydrochloric  acid  a  body  similar  to  urobilin  is 
formed.  As  an  oxidation  product  of  haematin  in  glacial  acetic  acid  with 
potassium  bichromate  or  chromium  trioxide,  Kuster  ^  obtained  the 
imide  of  the  tribasic  haematinic  acid,  C8HqN04,  which  is  also  produced  on 
theoxidation  of  haematoporphyrin  and  bilirubin. 

The  imide  of  the  tribasic  hsematinic  acid,  which  is  a  derivative  of  maleic  acid 

CO 
and  probably  has  the  formula  C5H7(COOH)  <r^r\>  NH,  is  readily  transformed  into 

the  anhydride  of  the  tribasic  hsematinic  acid,  CsHgOj,  having  the  probable  formula 
CH3.C.CO 

1 1        >  O.     On  heating   the   imide   with  alcoholic  ammonia  to 
C00H.CH2.CH,.C.C0 

130°  C.  it  spHts  off  carbon  dioxide,  and  the  imide  of  the  bibasic  hsematinic  acid, 
C7H9XO2,  is  obtained.     From  this  imide  on  saponification  with  baryta-water  we 

'  Zeitschr.  f.  analyt    Cfiem.,  43. 

'  Hoppe-Seyler,  Med.-chem.  Untersuch.,  p.  525;  Cloetta,  Arch.  f.  exp.  Path.  u. 
Pharm.,  3(5;   Rosenfeld,  ibid.,  40. 

^  Beitriige  zur  Kenntnis  des  Hiimatins,  Tubingen,  1896;  Ber.  d.  d.  chem. 
Gesellsch.,  2",  30,  32,  and  35;  Annal.  d.  '  hem.  u.  Pharm.,  315,  and  Zeitschr.  f.  physiol. 
Chem.,  28,  40,  and  44. 


HiEMATIN   AND  H^MIN.  211 

obtain  the  barium  salt  of  an  acid  whose  anhydride  is  methyl-ethyl  male'ic-acid 

C^Hj.C.CO 
anhydride,  ||       >0. 

CH3.  C.CO 
The  yield  of  haematinic  acids  is  so  great  that  Kuster  considers  that  at  least 
three  if  not  four  molecules  CgHgNO^  are  formed  from  one  haematin  molecule.  On 
heating  haematinic  acid  ester  with  alcoholic  ammonia  in  a  tube  to  130°  Kvster 
obtained  a  colored  product  whose  bluish-violet  aqueous  solution  gave  a  spectrum 
with  two  bands  which  in  position  were  similar  to  the  oxyhaemoglobin  spectrum. 

Haematin  is  amorphous,  dark  broun  or  bluish  black.  It  may  be  heated 
to  180°  C.  without  decomposition ;  on  burning  it  leaves  a  residue  consisting 
of  iron  oxide.  It  is  insoluble  in  water,  dilute  acids,  alcohol,  ether,  and 
chloroform,  but  it  dissolves  slightly  in  warm  glacial  acetic  acid.  Haematin 
dissolves  in  acidified  alcohol  or  ether.  It  easily  dissolves  in  alkalies,  even 
when  ver\'  dilute.  The  alkaline  solutions  are  dichroitic;  in  thick  layers 
they  appear  red  by  transmitted  light  and  in  thin  layers  greenish.  The 
alkaline  solutions  are  precipitated  by  lime-  and  baryta-water,  as  also  by 
solutions  of  neutral  salts  of  the  alkaline  earths.  The  acid  solutions  are 
alwa5's  browii. 

An  acid  haematin  solution  absorbs  the  red  part  of  the  spectnmi  only 
slightly  and  the  violet  parts  strongly.  The  solution  shows  a  rather  sharply 
defined  band  between  C  and  D,  whose  position  may  change  with  the  variety 
of  acid  used  as  a  solvent.  Between  D  and  F  a  second,  much  broader,  less 
shar^Dly  defined  band  occurs,  which  by  proper  dilution  of  the  liquid  is  con- 
verted into  two  bands.  The  one  between  h  and  F,  13'ing  near  F,  is  darker 
ai  d  broader;  the  other,  between  D  and  E,  lying  near  E,  is  lighter  and  nar- 
rower. Also  by  proper  dilution  a  fourth  ven,-  faint  band  is  observed  be- 
tween T>  and  E,  lying  near  D.  Haematin  may  thus  in  acid  solution  show- 
four  absorption-bands;  ordinarily  one  sees  distinctly  onty  the  bands  be- 
tween C  and  D  and  the  broad,  dark  band — or  the  two  bands — between  D 
and  F.  In  alkaline  solution  haematin  shows  a  broad  absorption-band, 
which  lies  in  greatest  part  between  C  and  D,  but  reaches  a  little  over  the 
line  D  towards  the  right  in  the  space  between  D  and  E.  As  the  position 
of  the  haematin  bands  in  the  spectrum  is  quite  variable,  the  exact  wave- 
lengths corresponding  thereto  cannot  be  given  exactly. 

Haemin,  H.emin  Crystals,  or  Teichmaxn's  Crystals.  Hsemin  is 
the  hydrochloric-acid  ester  of  haematin  and  is  the  startmg-point  m  the 
preparation  of  the  latter. 

The  statements  as  to  the  composition  of  ha^min  are  just  as  variable 
as  those  for  haematin,  which  is  partly  due  to  the  fact,  as  shown  by  Nexcki 
and  Zaleski,  that  the  haematin,  which  contains  two  hydroxyls  in  the 
molecule,  may  form  ethers  with  acids  and  alkyl  radicals,  which  also  yield 
addition  products  with  indifferent  compounds.  Thus  the  hsemin  prepared 
according  to  Xexcki  and  Sieber's  method  contains  amyl  alcohol. 
Schalfejeff's  haemin,  having  the  formula  C34H,33X4Fe04Cl,  is  supposed 


212  THE  BLOOD. 

to  contain  an  acetyl  group  and  hence  is  called  acethferain.  Morner's 
heemin,  C35H35N4Fe04Cl,  is  considered  as  a  monoethyl  ether  of  acethsemin. 
The  investigations  of  Zaleski,  Hetper  and  INIarchlewski,  K.  Morner, 
and  especially  those  of  Kl'Ster  have  given  explanations  of  these  conditions. 
The  so-called  acetha?min  does  not  contain  any  acetic-acid  radical,  hence 
its  name  is  incorrect.  Kuster,  by  a  new  method  of  purification  and  recrj-s- 
tahization,  has  shown  that  the  older  various  kinds  of  hsemins  were  not 
chemical  individuals  and  that  we  have  only  one  haemin.  This  view  is  now 
accepted  by  Morner  and  most  of  the  other  investigators,  and  the  formula 
C34H3304N4FeCl  is  now  given  to  hsemin.  Piettre  and  Vila  ^  dispute  this 
formula,  and  they  claim  to  have  prepared  chlorine-free  hsemin  from  pure 
cr}'stalline  oxy haemoglobin. 

Hsemin  crj^stals  form  in  large  masses  a  bluish-black  powder,  but  are  so 
small  that  they  can  only  be  seen  by  aid  of  the  microscope.  They  consist 
of  dark-brown  or  nearly  brownish-black  long,  rhombic,  or  spool-like 
crj'stals,  isolated  or  grouped  as  crosses,  rosettes,  or  stellar  forms.  Cubical 
crystals  may  also  occur,  according  to  Cloetta.  They  are  insoluble  in 
water,  dilute  acids  at  the  normal  temperature,  alcohol,  ether,  and  chloro- 
form. They  are  slightly  soluble  in  glacial  acetic  acid  with  heat.  They 
dissolve  in  acidified  alcohol,  as  also  in  dilute  caustic  alkalies  or  carbonates; 
and  in  the  last  case  they  form,  besides  alkali  chlorides,  soluble  hsematin 
alkali,  from  which  the  hsematin  may  be  precipitated  by  an  acid. 

On  shaking  with  cold  aniline  and  treating  first  with  acetic  acid  and 
then  with  ether,  Kuster  obtained  a  product,  dehydrochloride  haemin, 
which  was  poor  in  the  elements  of  hydrochloric  acid  and  which  again  took 
up  HCl  and  was  converted  into  hsemin.  By  the  action  of  boiling  aniline, 
hydrogen  is  driven  out  and  a  combination  with  aniline,  without  loss  of 
iron,  takes  place. 

The  principle  of  the  preparation  of  haemin  crystals  in  large  quantities 
is  as  follows:  The  washed  sediment  from  the  blood-corpuscles  is  coagu- 
lated vnth.  alcohol  or  by  boiling  after  dilution  with  water  and  the  careful 
addition  of  acid.  The  strongly  pressed  but  not  dry  mass  is  rubbed  with 
90-95  per  cent  alcohol  which  has  been  previously  treated  with  oxalic  acid 
or  T^-1  per  cent  concentrated  sulphuric  acid,  and  this  is  allowed  to  stand 
several  hours  at  the  temperature  of  the  room.  The  filtrate  is  warmed  to 
about  70°  C,  treated  with  hydrochloric  add  (for  each  litre  of  filtrate  add 
10  c.c.  25  per  cent  hydrochloric  acid  diluted  with  alcohol — IMorner), 
and  allowed  to  stand  in  the  cold.    The  ciystals,  which  separate  in  one 

^Nencki  and  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  30;  Nencki  and  Sieber,  Arch. 
f.  exp.  Path.  u.  Pharm.,  18  and  20,  and  Ber.  d.  d.  chem.  Gesellsch.,  18;  Schalfejeff  with 
Nencki  and  Zaleski,  1.  c;  Bialobrzeski,  Arch,  des  scienc.  biol.  de  St.  Petersbourg,  o; 
K.  Morner,  Nord.  Med.  Arkiv,  Festband,  1897,  Nos.  1  and  26,  and  Zeitschr.  f.  physiol. 
Chem.,  41;  Zaleski,  ifeid.,  37;  Hetper  and  Marchlewski,  i?jxi/.,  41  and  42;  Kuster ,  ibid., 
40;  Piettre  and  Vila,  Compt.  rend.,  141,  p.  734. 


I^MATOPORPHYRIN.  213 

or  two  days,  are  first  washed  with  alcohol  and  then  with  water.  For  pav- 
ticulars  as  to  the  various  methods  of  preparation  and  purification  we  refer 
the  reader  to  the  above-cited  works  of  Nencki  and  Sieber,  Cloetta, 
MoRXER,  Rosexfeld,  Nexcki  and  Zaleski  (Schalfejeff)  ,  and  especially 

to    KuSTER.l 

Hffimatin  is  obtained  on  dissolving  the  ha?min  crystals  in  ver\-  dilute 
caustic  alkali  and  precipitating  with  an  acid. 

In  preparmg  ha^min  crystals  in  small  quantities  proceed  m  the  follo-uing 
manner:  The  blood  is  dried  after  the  addition  of  a  small  quantity  of  com- 
mon salt,  or  the  dried  blood  may  be  rubbed  with  a  trace  of  the  same. 
The  drj^  powder  is  placed  on  a  microscope  slide,  moistened  with  glacial 
acetic  acid,  and  then  covered  with  the  cover-glass.  Add,  by  means  of  a 
glass  rod,  more  glacial  acetic  acid  by  applying  the  drop  at  the  edge  of  the 
cover-glass  until  the  space  between  the  slide  and  the  cover-glass  is  full. 
Now  warm  over  a  ver\'  small  flame,  with  the  precaution  that  the  acetic 
acid  does  not  boil  and  pass  with  the  powder  from  under  the  cover-glass. 
If  no  crj'stals  appear  after  the  first  warming  and  cooling,  warm  again,  and 
if  necessary  add  some  more  acetic  acid.  After  cooling,  if  the  experiment 
has  been  properly  performed,  a  number  of  dark-browii  or  nearly  black 
hajmin  crystals  of  varying  forms  will  be  seen. 

In  regard  to  the  preparation  of  iodohaematin  and  the  use  of  the  same 
for  the  detection  of  blood  we  must  refer  to  Strzyzowski's  communication  .^ 

By  the  action  of  acids  upon  hsemochromogen,  hsematin,  or  haemin  a 
new  iron-free  pigment,  which  was  first  closely  studied  by  Hoppe-Seyler 
and  called  hcBmatoporphi/rin,  is  produced.  According  to  the  method  of 
preparation  hsematoporphyrins  having  different  solubilities  and  whose 
relationship  to  each  other  is  not  perfectly  clear  are  produced,  but  all  show 
the  same  characteristic  absorption-spectmm.  The  best-studied  haemato- 
porphyrin  is  the  one  obtamed  according  to  Nexcki  and  Sieber's  method, 
by  the  action  of  glacial  acetic  acid  saturated  with  hydrobromic  acid  upon 
hsemm  cr\'stals,  best  at  the  temperature  of  the  body  (Nexcki  and 
Zaleski  ^) . 

Haematoporphyrin,  C16H18N2O3,  or  C34H38N4O6  according  to  Zaleski.* 
Tliis  pigment,  according  to  Mac^Iuxx,^  occurs  as  a  physiological  pigment 
in  certain  animals.  It  occurs,  as  show7i  by  Garrod  and  Saillet,  as  a 
normal  constituent,  although  only  as  traces,  of  human  urine.  It  occurs 
in  greater  quantities  in  human  urme  after  the  use  of  sulphonal  (see 
Chapter  XV). 

The  formation  of  hseraatoporphyrin  from  hsematin  can  be  expressed 


'  Zeitsclir.  f.  physiol.  Chem.,  40. 

2  Therapeut.  Monatshefte,  1901  and  1902. 

'  Hoppe-Seyler,  Med.-chem.  Untersuch./  528;  Nencki  and  Sieber,  Monatshefte  f. 
Chem.,  9,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  18,  20,  and  24;  Nencki  and  Zaleski, 
Zeitschr.  f.  physiol.  Chem.,  30. 

^Zeitschr.  f.  physiol,  Chem.,  37,  54. 

*  Joum.  of  Physiol.,  7. 


214  THE  BLOOD. 

by  the  following  equation  if  we  start  with  the  above  formula  for  hsemin 
and  Zaleski's  formula  for  hsematoporphyrin : 

C34H33N404FeCl  +  2HBr  +  2H2O  =  C34H38N4O6  +  FeBro  +  HCl. 

On  heating  ha3matoporphyrin  it  generates  an  odor  of  pyrrol.  On  oxidation 
with  bichromate  and  glacial  acetic  acid  it  yields  hsematinic  acid  (see  page 
210).  A  pigment  closely  allied  to  the  urinary  pigment  urobilin  has  been 
obtained  by  the  action  of  reducing  substances  on  hsematoporphyrin 
(Hoppe-Seyler,  Nencki  and  Sieber,  Le  Nobel,  MacMunn).  On  the 
administration  of  haematoporpyhrin  to  rabbits,  Nencki  and  Rotschy  ^ 
observed  that  a  part  was  reduced  to  a  substance  similar  to  urobilin. 

Of  especial  interest  are  the  recent  investigations  of  Nencki,  ^Iarch- 
LEWSKi,  and  Zaleski^  upon  the  reduction  products  of  hsematoporphyrin 
and  their  relationship  to  the  chlorophyll  derivatives.  By  the  action  of 
glacial  acetic  acid  containing  HI  and  of  iodophosphonium  upon  hsemin  or 
baemochromogen  Nencki  and  Zaleski  obtained  a  markedly  characteristic 
pigment,  meso porphyrin,  having  the  formula  C16H18N2O2,  or,  according 
to  Zaleski,^  C34H38N4O4,  and  which  stands  in  a  certain  measure  between 
hsematoporphyrin,  C16H18N2O3,  and  the  chlorophyll  derivative  phyllo- 
porphyrin,  C16H18N2O,  which  is  very  similar  to  hsematoporphyrin.  By 
the  action  of  the  same  reducing  agent  upon  hsemin  or  baemochromogen, 
but  under  other  conditions,  we  obtain  hcBmopyrrol,  CsHisN,  a  colorless 
oil,  which  in  the  air  gradually  changes  into  urobilin.  Haemopyrrol  is 
produced  by  the  action  of  the  same  reducing  agents  upon  the  chlorophyll 
derivative  phyllocyanin  (Nencki  and  Marchlewski),  which,  as  above 
remarked,  shows  a  close  relationship  between  the  blood-pigment  and 
chlorophyll. 

According  to  Nencki  and   Zaleski  haemopyrrol  is  probably  3-methyI-4-n- 
CH3 — C — C — C3H7 

propylpyrrol,  HC    CH.  Kuster  obtained  an  imide  from  hsemopyrrol  on 


NH 
oxidation  which  was  probably  a  derivative  of  methylpropylmaleic  acid.  As 
hsematinic  acid  's  undoubtedly  a  maleic-acid  derivative,  it  was  of  interest  to 
prove  the  correctness  of  the  above  formula  of  hsemopyrrol,  and  with  this  purpose 
in  view  Kuster  and  Haas  ^  have  compared  the  synthetically  prepared  imide 
of  methylpropylmaleic  acid  with  the  imide  obtained  from  haemopyrrol.  The  two 
bodies  were  not  identical,  therefore  the  above  constitutional  formula  is  ques- 

^  Hoppe-Seyler,  1.  c,  523;    Le  Nobel,  Pfl  ger's  Arch.,  40;    MacMunn,  Proc.  Roy. 
Soc,  30,  and  Journ.  of  Physiol.,  10;  Nencki  and  Rotschy,  Monatshefte  f.  Chem.,  10. 
2  See  foot-note  1,  p.  197. 
'  Zeitschr.  f.  physiol.  Chem.,  37. 
*  Ber.  d.  d.  chem.  Gesellsch.,  3". 


ILEMATOPORPHYRIX.  215 

tioned.  The  attempt  of  Buraczewski  and  Marchlewski  ^  to  prepare  hsemo- 
pyrrol  artificially  from  methylpropylmaleic-acid  imide  yielded  a  product  similar 
to  hsemopyrrol,  which  on  oxidation  in  the  air  did  not  yield  a  typical  uro- 
bilin but  at  least  a  substance  closely  related  thereto.  The  assumption  that 
hsemopyrrol  is  a  pyrrol  derivative  is  best  borne  out  by  the  property  which  hsemo- 
pyrrol  has  of  reacting  with  diazonium  compounds  with  the  formation  of  azo 
pigments  (Goldmaxn,  Marchlewski,  Hetper  ■). 

HEBmatoporphyrin  is,  according  to  Nencki  and  Sieber,  isomeric  with 
the  bile-pigment  bilirubin,  and  like  this  latter  gives  a  play  of  colors — green, 
blue,  and  yellow — when  treated  with  fuming  nitric  acid.  The  hydrochloric- 
acid  compound  crj'stallizes  in  long  bro^^■nish-red  needles.  If  the  solution 
in  hydrochloric  acid  is  nearly  neutralized  with  caustic  soda  and  then  treated 
with  sodium  acetate,  the  pigment  separates  ovit  as  amorphous,  brown 
fiakes  not  readily  soluble  in  amyl  alcohol,  ether,  and  chloroform,  but  readily 
soluble  in  ethyl  alcohol,  alkalies,  and  dilute  mineral  acids.  The  com- 
pound with  sodium  crj^stallizes  as  small  tufts  of  brown  cr}-stals.  The  acid 
alcoholic  solutions  have  a  beautiful  purple  color,  which  becomes  \iolet- 
blue  on  the  addition  of  large  quantities  of  acid.  The  alkaline  solution  has 
a  beautiful  red  color,  especially  when  not  too  much  alkali  is  present. 

An  alcoholic  solution  of  hsematoporphy rin ,  acidulated  with  hydrochloric 
or  sulphuric  acid,  shows  two  absorption-bands,  one  of  which  is  fainter  and 
narrow'er  and  lies  betw^een  C  and  D,  near  D.  The  other  is  much  darker, 
sharper,  and  broader,  and  lies  midway  betw'een  D  and  E.  An  absorption 
extends  from  these  bands  towards  the  red,  terminating  with  a  dark  edge, 
which  may  be  considered  as  a  third  band  between  the  other  two. 

A  dilute  alkaline  solution  shows  four  bands,  namely,  a  band  between 
C  and  D;  a  second,  broader  band  surrovmding  D  and  with  the  greater  part 
between  D  and  E;  a  third  between  D  and  E,  nearly  at  E;  and  lastly,  a 
fourth  broad  and  dark  band  between  h  and  F.  On  the  addition  of  an 
alkaline  zinc-chloride  solution  the  spectrum  changes  more  or  less  rapidly/^ 
and  finally  a  spectrum  is  obtained  with  only  tW'O  bands,  one  of  which 
surrounds  D  and  the  other  lies  between  D  and  E.  If  an  acid  haematopor- 
phyrin  solution  is  shaken  wdth  chloroform,  a  part  of  the  pigment  is  taken 
up  by  the  chloroform,  and  this  solution  often  shows  a  five-banded  spectrum 
with  two  bands  between  C  and  D.  The  position  of  the  hiematoporphyrin 
bands  in  the  spectrum  differs  with  the  various  methods  of  preparation 
and  other  conditions,  so  that  they  do  not  correspond  to  the  same  w^ave- 
length.  These  facts  coincide  well  with  the  recent  investigations  of  A. 
ScHULz;'*  according  to  which  the  appearance  of  the  spectiaim  is  not  only 

^  Zeitschr.  f.  physiol.  Chem., -13. 

2  Ibid.,  43  and  45. 

^  See  Hammarsten,  Skand.  Arch.  f.  Physiol.,  3,  and  Garrod.  Journ.  of  Physiol.,  13. 

'Arch.  f.  (Anat.  u.)  Physiol.,  1904,  Suppl. 


216  THE  BLOOD. 

dependent  upon  the  reaction  but  also  upon  the  character  of  the  solvent 
and  the  method  of  preparation. 

In  regard  to  the  preparation  of  hcematoporphyrin,  see  Hoppe-Seyler- 
Thierfelder's  Handbuch,  7.  Aufl..  and  the  works  cited  on  page  213. 

Haematinogen  is  a  ferruginous  pigment  so  named  by  Freund,'  which  he 
obtained  by  carefully  extracting  blood  with  alcohol  containing  hydrochloric  acid. 
It  is  closely  related  to  hoematin,  but  is  not  sufficiently  characteristic  and  is  not 
considered  as  a  cleavage  product. 

A  question  of  great  interest  is  whether  it  is  possible  to  produce  the 
blood-pigment  from  its  cleavage  products.  In  this  regard  certain  recent 
investigations  are  interesting.  Zaleski  obtained  from  mesoporphyrin 
hydrochloride  dissolved  in  80  per  cent  acetic  acid  saturated  with  NaCl 
and  heated  to  50-70°,  a  hsemin-like  pigment  by  the  addition  of  a  solu- 
tion of  iron  in  acetic  acid,  and  this  pigment  had  a  spectrum  in  acid 
solution  very  similar  to  that  of  haematin,  although  not  identical  with  it. 
Zaleski  considers  this  pigment  as  a  hydrogenized  hsemin.  A  regeneration 
of  hsemin  from  hoematoporphyrin  has  been  performed  by  Laidlaw.  If 
hsematoporphyrin  is  dissolved  in  dilute  ammonia  and  warmed  with 
Stokes'  solution  and  hydrazine  hydrate,  iron  is  taken  up  again  and  haemo- 
chromogen  is  produced,  which  is  changed  into  haematin  by  shaking  with 
air.  According  to  Ham  and  Balean,^  it  is  possible  to  produce  haemoglo- 
bin from  haemochromogen  and  globin,  and  it  is  indeed  possible  that  othor 
proteins  can  replace  globin  in  this  formation. 

Haematoidin,  thus  called  by  Virchow,  is  a  pigment  which  crystallizes 
in  orange-colored  rhomlDic  plates,  and  which  occurs  in  old  blood  extrav^a- 
tions,  and  whose  origin  from  the  blood-coloring  matters  seems  to  be  estab- 
lished (Langhans,  Cordua,  Quincke,  and  others  2).  A  solution  of  haema- 
toidin shows  no  absorption-bands,  but  only  a  strong  absorption  from  the 
\'iolet  to  the  green  (E wald  ^) .  According  to  most  observers,  haematoidin 
is  identical  with  the  bile-pigment  bilimbin.  It  is  not  identical  with  the 
cr}\stallizable  lutein  from  the  corpora  lutea  of  the  ovaries  of  the  cow 
(Piccolo  and  Lieben,^  Kijhne  and  Ewald). 

In  the  detection  of  the  above-described  blood-coloring  matters  the 
spectroscope  is  the  only  entirely  trustworthy  means  of  investigation.  If 
it  is  only  necessary  to  test  for  blood  in  general  and  not  to  determine 
definitely    whether   the    coloring-matter   is   haemoglobin,    methsemoglobin , 

*  Wien.  kiln.  Wochenschr.,  1903. 

^  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  43;  Laidlaw,  Journ.  of  Physiol.,  31;  Ham. 
and  Balean,  ibid.,  32. 

'  A  comprehensive  review  of  the  literature  pertaining  to  haematoidin  may  be  found 
in  Stadelmann,  Der  Icterus,  etc.,  Stuttgart,  1891,  pp.  3  and  45. 

*  Zeitschr.  f.  Biologic,  22,  475. 

"  Cit.  from  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4.  Aufl,,  1878. 


UANTITATIVE    ESTIMATION    OF   BLOOD   PIGMENTS.        217 

or  hgematin,  then  the  ii reparation  of  hsemin  crv'stals  is  an  absokitely  positive 
test.  The  reader  is  referred  to  more  extended  text-books  for  more  exact 
methods  for  the  detection  of  Ijlood  in  chemico-legal  cases,  and  it  is  perhaps 
sufficient  to  give  here  the  chief  points  of  the  investigation. 

If  spots  on  clothes,  Hnen,  wood,  etc.,  are  to  be  tested  for  the  presence 
of  blood,  it  is  best,  when  possible,  to  scratch  or  shave  off  as  much  as 
possible,  rub  with  common  salt,  and  from  this  prepare  the  hsemin  crystals. 
On  obtaining  positive  results  the  presence  of  blood  is  not  to  be  doubted. 
When  sufficient  material  is  not  obtained  by  the  above  meaiis,  soak  the 
spot  with  a  few  drops  of  water  in  a  watch-crystal.  If  a  colored  solution 
is  thus  obtained,  then  remove  the  fibres,  wood-shavings,  and  the  like  as 
far  as  possible,  and  allow  the  solution  to  dry  in  the  watch-glass.  The 
dried  residue  may  be  partly  used  for  the  spectroscopic  test  directly,  and 
part  may  be  employed  in  the  preparation  of  the  hsemin  crystals.  It  may 
also  be  used  to  detect  ha^mochromogen  in  alkaline  solution  after  previous 
treatment  with  alkali  and  the  addition  of  reducing  substances. 

If  a  colorless  solution  is  obtained  after  soaking  with  water,  or  if  the  spots 
are  on  rusty  iron,  then  digest  with  a  little  dilute  alkali  (5  p.  m.).  In  the 
presence  of  blood  the  solution  gives,  after  neutralization  with  hydro- 
chloric acid  and  drjing,  a  residue  which  may  give  the  hsemin  crystals  with 
glacial  acetic  acid.  Another  part  of  the  alkaline  solution  shows,  after 
thQ  addition  of  Stokes'  reduction  fluid,  the  absorption-bands  of  hsemo- 
chromogen  in  alkaline  solution. ^ 

Th3  methods  proposed  for  the  quantitative  estimation  of  the  blood- 
coloring  matters  are  partly  chemical  and  partly  physical. 

Among  the  chemical  methods  to  be  mentioned  is  the  incineration  of  the 
blood  and  the  determination  of  the  amount  of  iron  contained  in  the  ash,  from 
which  the  amount  of  haemoglobin  may  be  calculated.  Jolles  ^  has  recently  sug- 
gested a  clinical  method  based  on  this  procedure. 

The  physical  methods  consist  either  of  coloriraetric  or  of  spectroscopic 
investigations. 

The  principle  of  Hoppe-Seyler's  colorimetric  method  is  that  a  measured 
quantity  of  blood  is  diluted  with  an  exactly  measured  quantity  of  water 
until  the  diluted  blood  solution  has  the  same  color  as  a  pure  oxyhsemo- 
globin  solution  of  a  known  strength.  The  amount  of  coloring-matter 
present  in  the  undiluted  blood  may  be  easily  calculated  from  the  degree  of 
dilution.  In  the  colorimetric  testing  we  use  a  glass  vessel  with  parallel 
sides  containing  a  layer  of  liquid  1  cm.  thick  (Hoppe-Seyler's  haematinom- 
eter).  The  use  of  Hoppe-Seyler's  colorimetric  double  pipette  is  more 
advantageous.  Other  good  forms  of  apparatus  have  been  constructed  by 
GiACOSA  and  Zangermeister.^  Instead  of  an  oxyhsemoglobin  solution  we 
row  generally  use  a  carbon-monoxide  hsemoglobin  solution  as  a  standard 

'On  the  use  of  color  reactions  for  the  detection  of  blood,  see  O.  and  R.  Adler, 
Zeitschr.  f.  physiol.  Chem.,  41,  and  Schumm  and  Westphal,  ibid.,  46. 

^  Pfliiger's  Arch.,  65;  Monatshefte  f.  Chem.,  17.  See  also  Oerum,  Zeitschr.  f. 
anal.  Chem.,  48,  and  the  works  cited  in  Maly's  Jahresber,,  33. 

'  F.  Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  16;  G.  Hoppe-Seyler,  ibid.,  21; 
Winternitz,  ibid.,  Giacosa,  Maly's  Jahresber.,  26;  Zangermeister,  Zeitsclir.  f.  Bio'.o- 
gie.  33. 


218  THE  BLOOD. 

liquid  because  it  may  be  kept  for  a  long  time.     The  blood  solution  in  this 

case  is  saturated  with  carbon  monoxide.^ 

The  quantitative  estimation  of  the  blood -coloring  matter?  by  means  of 

the  spectroscope  may  be  done  in  different  ways,  but  at  the  present  time 

the  spedrophotometric  method  is  chiefly  used,  and  this  seems  to  be  the 

most  reliable.     This  method  is  based  on  the  fact  that  the  extinction  coeflB- 

cient  of  a  colored  hquid  for  a  certain  region  of  the  spectrum  is  directly 

proportional  to  the  concentration,  so  that  C:E=C\:Ei,  when  C   and  C'l 

represent  the  different  concentrations  and  E  and  Ei   the  corresponding 

C     C 
coefl&cients  of  extinction.    From  the  equation  TT  =  Tr>  it  follows  that  for 

one  and  the  same  pigment  this  relation,  which  is  called  the  absorption 
ratio,  must  be  constant.  If  the  absorption  ratio  is  represented  by  A,  the 
determined  extinction  coefficient  by  E,  and  the  concentration  (the  amoimt 
of  coloring-matter  in  grams  in  1  c.c.)  b}'  C,  then  C=A  .  E. 

Different  forms  of  apparatus  have  been  constructed  (Yierordt  and 
HuFXER  2)  for  the  determination  of  the  extinction  coefficient,  which  is  equal 
to  the  negative  logarithm  of  those  rays  of  light  which  remain  after  the  pas- 
sage of  the  licht  through  a  layer  1  cm.  thick  of  an  absorbing  liquid.  In 
regard  to  this  apparatus  the  reader  is  referred  to  other  text-books. 

For  purposes  of  control  the  extinction  coefficients  are  determined  in  two/fif- 
ferent  regions  of  the  spectrum.  Hufner  has  selected  (a)  the  region  between  the 
two  absorption-bands  of  oxyhaemoglobin,  especially  between  the  wave-lengths 
5.54  .«,«  and  .565  ,«,«,  and  (h)  the  region  between  the  two  bands,  especially  the  inter- 
val between  the  wave-lengths  .5.31. .5  ,«,«  and  .542..5  ,«,«.  The  constants  or  the 
absorption  ratio  for  these  two  regions  of  the  spectrum  are  designated  by  Hufxer 
by  A  and  A'.  Before  the  determination  the  blood  must  be  diluted  with  water, 
and  if  the  proportion  of  dilution  of  the  blood  be  represented  by  V,  then  the  fon- 
centratiou  or  the  amount  of  coloring-matter  in  100  parts  of  the  undiluted  blood  is 

C  =  100.r  .,4  .£:and 
C  =  100.r  ..4'.£'. 

The  absorption  ratio  or  the  constants  in  the  two  above-mentioned  regions 
of  the  spectrum  have  been  determined  for  oxyhaemoglobin,  haemoglobin,  carbon 
monoxide  haemoglobin,  and  methaemoglobin,  as  follows: 

Oxvhaemoglobin Ao  =0.002070  and  A'o  =0.001.312 

Haemoglobin Ar  =0.001.3.54  and  A'r  =0.001778 

Carbon-monoxide  haemoglobin.  .  ..4c  =0.001.383  and  A'c  =0.001263 
Methaemoglobin .4 „^^  =0.002077  and  A  'm  =0.0017.54 

The  quantity  of  each  coloring-matter  may  be  determined  in  a  mixture  of 
two  blood-coloring  matters  by  this  method;  this  is  of  special  importance  in 
the  determination  of  the  quantity  of  oxyhaemoglobin  and  haemoglobin  present 
in  Vjlood  at  the  same  time. 

In  order  to  facilitate  these  determinations,  Hufxer  '  has  worked  out  tables 
which  give  the  relation  between  the  two  pigments  existing  in  a  solution  contain- 

'  See  Haldane,  Joum.  of  Physiol.,  26. 

^  See  Vierordt,  Die  Anwendung  des  Spektralapparates  zu  Photometrie,  etc.  (T  bin- 
gen,  1873J,  and  H  fner,  Arch.  f.  (.\nat.  u.)  Fhysiol.,  1894,  and  Zeitschr.  f.  physiol. 
Chem..  3;  v   Noorden.  jbtV/.,  4:  Otto.  Pfl  ger's  Arch..  31  and  36. 

-Arch.  f.  (.\nat.  u.)  Physiol.,  1900. 


RED    BLOOD  CORPUSCLES.  219 

ing  oxyhi3emoglobin  and  another  pigment  (haemoglobin,  methsemoglobin,  or  carbon- 
monoxide  haemoglobin),  and  thus  allowing  of  the  calculation  of  the  absolute  quan- 
tity of  each  pigment. 

Among  the  many  apparatuses  constructed  for  clmical  purposes  for  the 
quantitative  estimation  of  hix-moglobin,  Fleischl's  hcemometer,  which  has 
undergone  numerous  modifications,  HExocQUf:'s  hcematoscope,  and  Sahli's 
hcemometer  are  to  be  specially  mentioned.  In  regard  to  these  apparatuses, 
see  V.  Jaksch,  Klinische  Diagnostik  innerer  Krankheiten,  4.  Auflage,  1897, 
and  Jaquet,  Korresp.-Blatt  f.  Schweiz.  Aerzte,  1897;  Gartner,  Miinchener 
med.  Wochenschr.,  1901,  and  H.  Sahli,  Diagnostic  Methods,  Philadelphia, 
1905. 

Many  other  pigments  are  found  besides  the  often-occurring  haemoglobin 
in  the  blood  of  invertebrates.  In  a  few  Arachnidae,  Crustacea,  Gasteropodse, 
and  Cephalopodse  a  body  analogous  to  haemoglobin,  containing  copper,  hcerno- 
cyanin,  has  been  found  by  Fredericq.  By  the  taking  up  of  loosely  bound  oxygen 
this  body  is  converted  into  blue  oxyhcemocyanin,  and  by  the  escape  of  the  oxygen 
l)ecomes  colorless  again.  According  to  Henze  1  gram  haemocyanin  combines 
-with  about  0.4  c.c.  oxygen.  It  is  crvstalline  and  has  the  following  composition: 
€5.3.66;  H7..3.3;  X  16.09;  S0.86;  Cu0..38;  0  21.67  per  cent.  On  hydrolytic 
cleavage  with  hydrochloric  acid  Henze  found  the  following  division  of  the  nitro- 
gen in  haemocyanin:  Of  the  total  nitrogen  .5.78  per  cent  was  spHt  off  as 
ammonia,  2.67  per  cent  as  humus  nitrogen,  27.65  per  cent  as  diamino  nitrogen, 
and  63.39  per  cent  as  monamino  nitrogen.  He  found  no  arginine  in  the  cleavage 
products,  but  could  detect  histidine,  lysine,  tyrosine,  and  glutamic  acid.  A  color- 
ing-matter called  chlorocruorin  by  Lankester  is  found  in  certain  Chsetopodse. 
Hcemerythrin,  so  called  by  Krukenberg  but  first  observed  by  Schw^\lbe,  is  a 
red  coloring-matter  from  certain  Gephyrea.  Besides  haemocyanin  we  find  in  the 
blood  of  certain  Crustacea  the  red  coloring-matter  tetronerythrin  (Halliburton), 
which  is  also  widely  spread  in  the  animal  kingdom.  Echinochrom,  so  named 
by  MacMunn,'  is  a  brown  coloring-matter  occurring  in  the  perivisceral  fluid  of 
a  variety  of  echinoderms. 

The  quantitative  constitution  of  the  red  hlood-corpusdes.  The  amount 
of  water  varies  in  different  varieties  of  blood-corpuscles  between  570-644 
p.  m.,  with  a  corresponding  amount,  430-356  p.  m.,  of  solids.  The  chief 
mass,  about  iV"¥o>  of  the  dried  substance  consists  of  hsemoglobin  (in  human 
xmd  mammalian  blood). 

According  to  the  analyses  of  Hoppe-Seyler  2  and  his  pupils,  the  red 
-corpuscles  contain  in  1000  parts  of  the  dried  substance: 

Haemoglobin  Protein  Lecithin  Cholesterin 

Human  blood 868-944  122-51  7.2-3.5  2.5 

Dog's         "     865  126  5.9  3.6 

Goose's      "     627  364  4.6  4.8 

Snake's     "    467  525 

Abderhalden  found  the  following  composition  for  the  blood-corpuscles 
from    the    domestic    animals   investigated   by   him:    Water,    591.9-644.3 

^  Fredericq,  Extrait  des  Bulletins  de  l.'Acad.  Roy.  de  Belgique  (2),  46,  1878;  Lan- 
kester,  Joum.  of  Anat.  and  Physiol.,  2  and  4;  Henze,  Zeitschr.  f.  physiol.  Chem.,  33 
and  43;  Krukenberg,  see  Vergl.  physiol.  Studien,  Reihe  1,  Abt.  3,  Heidelberg,  1880; 
Halliburton,  Journal  of  Physiol.,  6;  MacMunn,  Quart.  Journ.  Microsc.  Science,  1885. 

^  Med.-chem.  Untersuch.,  390  and  393. 


220  THE  BLOOD. 

p.  m.;  solids  408.1-335.7  p.  m.;  haemoglobin,  303.3-331.9  p.  m. ;  protein, 
5.32  (dog) -78.5  p.  m.  (sheep);  cholesterin,  0.388  (horse)-3.593  p.  m. 
(sheep);  and  lecithin,  2.296  (dog)-4.855  p.  m. 

Of  special  interest  is  the  varjdng  proportion  of  the  haemoglobin  to  the 
protein  in  the  nucleated  and  in  the  non-nucleated  blood-corpuscles.  These 
last  are  much  richer  in  haemoglobin  and  poorer  in  protein  than  the  others. 

The  amount  of  mineral  bodies  in  various  species  of  animals  is  different. 
According  to  Buxge  and  Abderhaldex  the  red  corpuscles  from  the  pig, 
horse,  and  rabbit  contain  no  soda,  while  those  from  man,  the  ox,  sheep, 
goat,  dog,  and  cat  are  relatively  rich  in  soda.  In  the  five  last-mentioned 
species  the  amount  of  soda  was  2.135-2.856  p.  m.  The  quantity  of  potash 
was  0.257  (dog)-0.744  p.  m.  (sheep).  In  the  horse,  pig,  and  rabbit  the 
quantity  of  potash  was  3.326  (horse)-5.229  p.  m.  (rabbit).  Human  blood- 
corpuscles  contain,  according  to  Waxach,  about  five  times  as  much  potash 
as  soda,  on  an  average  3.99  p.  m.  potash  and  0.75  p.  m.  soda.  The  nucleated 
er\^throcytes  of  the  frog,  toad,  and  turtle  contain,  according  to  Bottazzi 
and  Cappelli,^  also  considerably  more  potassium  than  sodium.  Lime  is 
claimed  to  be  absent  in  the  blood-corpuscles,  and  magnesia  occurs  only 
in  small  amounts:  0.016  (sheep)-0.150  p.  m.  (pig).  The  blood-corpuscles 
of  all  animals  investigated  contain  chlorine,  0.460-1.949  p.  m.  (both  in 
horse),  generalh'  1  to  2  p.  m.,  and  also  phosphoric  acid.  The  amount  of 
inorganic  phosphoric  acid  shows  great  variation:  0.275  (sheep)-1.916  p.  m. 
(horse).  All  of  the  above  figures  are  calculated  on  the  fresh,  moist  blood- 
corpuscles. 

By  quantitative  determinations  of  the  swelling  and  shrinking  of  the  cells 
under  the  influence  of  NaCl  solutions  of  various  concentration  or  of  serum  of 
various  dilutions,  Hamburger  has  attempted  to  determine  for  the  erythrocytes, 
as  well  as  the  leucocytes,  the  percentage  relationship  between  the  two  chief  con- 
stituents of  the  cells  (the  frame  and  the  intracellular  fluid).  He  found  that  the 
volume  of  the  frame-substance  for  both  varieties  of  blood-corpuscles  of  the  horse 
was  equal  to  53-56.1  per  cent.  The  volume  for  the  red  blood-corpuscles  was 
for  the  rabbit  48.7-51;  hen,  52.4-57.7,  and  for  the  frog,  72-76.4  per  cent. 
KoEPPE  has  raised  objections  to  these  determinations. - 

The  White  Blood-corpuscles  and  the  Blood-plates. 
The  White  Blood-corpuscles,  also  called  Leucocytes  or  Lymphoid 
Cells,  are  of  different  kinds,  and  ordinarily  we  differentiate  between  the 
small  forms  poor  in  protoplasm,  called  lymphocytes,  and  the  larger, 
granular,  often  more  nucleated  forms,  called  leucocytes.  The  polynuclear 
leucocytes  occur  in  greater  abundance  in  the  l^lood  than  the  lymphocytes. 


'  Bunge,  Zeitschr.  f.  Biologie,  12,  and  Abderhalden,  Zeitschr.  f.  physiol.  Chem.,  23 
and  25;  Wanach,  Maly's  Jahresber.,  18,  88;  Bottazzi  and  Cappelli,  Arch.  Ital.  de  Biolo- 
gie,  32. 

2  Hamburger,  Arch.  f.  (Anat.  u.)  Physiol.,  1898;  Koeppe,  ibid.,  1899  and  1900. 


LEUCOCYTES.  221 

In  human  and  mammalian  blood,  most  of  the  white  blood-corpuscles  are 
larger  than  the  red  blood-corpuscles.  They  have  also  a  lower  specific 
gra\ity  than  the  red  corpuscles,  move  in  the  circulating  blood  nearer  to 
the  walls  of  the  blood-vessels,  and  have  also  a  slower  motion. 

The  number  of  white  blood-corpuscles  varies  not  only  in  the  different 
b^ood -vessels,  but  also  under  different  physiological  conditions.  On  an 
average  there  is  only  1  white  corpuscle  for  3.50-500  red  corpuscles.  Accord- 
ing to  the  investigations  of  Alex.  Schmidt  ^  and  his  pupils,  the  leucocytes 
are  destroyed  in  great  part  on  the  discharge  of  the  blood  before  and  during 
coagulation,  so  that  discharged  blood  is  much  poorer  in  leucocytes  than 
the  circulating  blood.  The  correctness  of  this  statement  has  been  denied 
by  other  investigators. 

From  a  liistological  standpoint  we  generally,  as  above  indicated,  dis- 
criminate between  the  different  kinds  of  colorless  blood-corpuscles. 
Chemically  considered,  however,  there  is  no  knowTi  essential  difference 
between  them,  and  what  little  we  do  know  chemically  is  chiefly  in  con- 
nection with  the  leucocytes.  With  regard  to  their  importance  in  the 
coagulation  of  fibrin,  Alex.  Schmidt  and  his  pupils  distinguish  between 
the  leucocytes  which  are  destroyed  in  the  coagulation  and  those  which 
are  not.  The  last  mentioned  give  with  alkalies  or  common-salt  solutions 
a  slim}'  mass;  the  first  do  not  show  such  behavior. 

The  protoplasm  of  the  leucocytes  has  during  life  amoeboid  movements 
which  S3rve  partly  to  make  possible  the  wandering  of  the  cells,  and  partly 
to  aid  in  the  absorption  of  smaller  grains  or  foreign  bodies.  On  these  grounds 
the  occurrence  of  myosin  in  them  has  been  admitted  even  without  any 
special  proof  thereof.  Alex.  Schmidt  claims  to  have  found  serglobulin  in 
equine-blood  leucocytes  which  have  been  washed  with  ice-cold  water. 
There  are  also  certain  leucocytes,  as  above  stated,  which  yield  a  slimy  mass 
when  treated  with  alkalies  or  NaCl  solutions,  which  seems  to  be  identical 
with  the  so-called  hyaline  substance  of  Ro^^DA  found  in  the  pus-cells.  On 
digesting  the  leucocytes  with  water,  a  solution  of  a  protein  body  is  obtained 
which  can  be  precipitated  by  acetic  acid  and  which  forms  the  chief  mass  of  the 
leucocytes.  This  substance,  which  is  undoubtedly  concerned  in  the  coagu- 
lation of  the  blood,  has  been  described  under  different  names  (see  Chapter 
V,  page  141),  and  consists,  cliiefly,  at  least,  of  nucleoproteid.  The  ordinary' 
view^  that  this  is  nucleohistone  does  not  seem  to  be  correct,  according  to  the 
recent  investigations  of  Baxg,^  and  further  proof  is  necessar}-. 

Glycogen,  as  prexiousl}'  stated,  is  found  in  the  leucocytes.  The  glyco- 
gen found  by  Huppert,  Czerxy,  Dastre.^  and  others  in  blood  and  lymph 

■  Pfliiger's  Arch.,  11.     Also  Fr.  Kriiger,  Arch-  f.  exp.  Path.  u.  Phann.,  51. 

^  I.  Bang,  Studier  over  Nukleoproteider,  Kristiania,  1902. 

"  Huppert.  Centralbl.  f  Physiol.,  6,  394;  Czerny,  Arch.  f.  exp.  Path.  u.  Pharm..  31; 


222  THE  BLOOD. 

probably  originated  from  the  leucocytes.  A  glucothionic  acid  has  been 
prepared  from  white  cells  by  Mandel  and  Levene.^  The  constituents  of 
the  leucocytes  are  the  same  as  the  constituents  of  the  cell  as  described  in 
Chapter  V. 

The  blood-plates  (Bizzozero),  hsematoblasts  (Hayem),  whose  nature, 
preformed  occurrence,  and  physiological  importance  have  been  much  ques- 
tioned, are  pale,  colorless,  gummy  disks,  round  or  somewhat  oval  in  shape 
and  generally  with  a  diameter  two  or  three  times  smaller  than  the  red 
blood-corpuscles.  By  the  action  of  different  reagents  the  blood-plates 
separate  into  two  substances,  one  of  which  is  homogeneous  and  non-refrac- 
tive, while  the  other  is  highly  refractive  and  granular.  Blood-plates 
readily  stick  together  and  attach  themselves  to  foreign  bodies. 

According  to  the  researches  of  Kossel  and  of  Lilienfeld  ^  the  blood- 
plates  consist  of  a  chemical  combination  between  protein  and  nuclein, 
and  hence  they  are  also  called  nuclein-plates  by  Lilienfeld,  and  are  con- 
sidered as  derivatives  of  the  cell  nucleus.  It  seems  certain  that  the  blood- 
plates  have  some  connection  with  the  coagulation  of  blood.  The  views  on 
this  question  and  especially  in  regard  to  the  manner  in  which  these  plates 
act  in  coagulation  are  unfortunately  very  divergent. 

m.    THE  BLOOD  AS  A  MIXTURE  OF  PLASMA  AND  BLOOD-CORPUSCLES. 

The  blood  in  itself  is  a  thick,  sticky,  light  or  dark  red  liquid,  opaque 
even  in  thm  layers,  having  a  salty  taste  and  a  faint  odor  differing  m  differ- 
ent kinds  of  animals.  On  the  addition  of  sulphuric  acid  to  the  blood  the 
odor  is  more  pronounced.  In  adult  human  beings  the  specific  gravity 
ranges  between  1.045  and  1.075.  It  has  an  average  of  1.058  for  growii 
men  and  a  little  less  for  women.  Lloyd  Jones  found  that  the  specific 
gra\aty  is  highest  at  birth  and  lowest  in  children  when  about  two  years  old 
and  in  pregnant  women.  The  determinations  of  Lloyd  Jones,  Hammer- 
SCHLAG,^  and  others  show  that  the  variation  of  the  specific  gravity,  depend- 
eai  upon  age  and  sex,  corresponds  to  the  variation  in  the  quantity  of 
haemoglobin. 

The  determination  of  the  specific  gravity  is  most  accurately  done  by 
means  of  the  pyknometer.     For  clinical  purposes,  where  only  small  amounts 

Dastre,  Compt.  rend.,  120,  and  Arch,  de  Physiol.  (5),  7-  See  also  Hirschberg,  Zeitschr. 
f.  klin.  Med.,  54. 

i  Biochem.  Zeitschr.,  4. 

*  In  regard  to  the  literature  of  the  blood-plates,  see  Lilienfeld,  Arch.  f.  (Anat.  u.) 
Physiol.,  1892,  and  "Leukocyten  und  Blutgerinnung,"  Verhandl.  d.  physiol.  Gesellsch. 
zu  Berhn,  1892;  and  also  Mosen,  Arch,  f  (Anat  u  )  Physiol.,  1893,  and  Maly's  Jahres- 
ber..  30  and  31. 

"Lloyd  Jones,  Journ.  of  Pliysiol.,  S;  Hammerschlag,  Wien.  klin.  Wochenschrift, 
1890,  and  Zeitsclir  f.  klin.  Med.,  20. 


ALKALINITY   OF  THE  BLOOD.  225 

are  available,  it  is  best  to  proceed  with  tiie  method  as  suggested  by  Ham- 
MERSCHLAG.  Prepare  a  mixture  of  chloroform  and  benzene  of  about  1.050 
sp.  gr.  and  add  a  drop  of  the  blood  to  this  mixture  If  the  drop  rises  to 
the  surface  then  add  benzene,  and  if  it  sinks  add  chloroform.  Continue 
this  until  the  drop  of  blood  suspends  itself  midway  and  then  determine 
the  specific  gravity  of  the  mixture  by  means  of  an  areometer.  This  method 
is  not  strictly  accurate  and  must  be  performed  quickly.  In  regard  to  the 
necessary  details  refer  to  Zuntz  and  A.  Levy.^ 

The  reaction  of  the  blood  is  alkaline  towards  litmus.  The  quantity  of 
alkali,  calculated  as  NaoCOs,  in  fresh,  non-defibrinated  blood  from  the 
dog,  horse,  and  man,  is,  according  to  Loewy,  4.93,  4.43,  and  5.95  p.  m.^ 
respectively.  According  to  Strauss  the  average  for  normal  human  blood 
may  be  calculated  as  about  4  3  p.  m,  Na2C03.  Quantities  below  3.3  p.  m.. 
and  above  5.3  p.  m.  are,  according  to  him,  to  be  considered  as  pathologicaL 
V.  Jaksch  found  the  quantity  of  alkali  in  man  to  vary  between  3.38  and 
3.90  p.  m.,  and  Brandenburg  found  3  p.  m.  NaOH(=3.98  p.  m.  Na2C03). 
The  alkaline  reaction  diminishes  outside  of  the  body,  and  indeed  the  more 
quickly  the  greater  the  original  alkalinity  of  the  blood.  This  depends  on 
the  formation  of  acid  in  the  blood,  in  which  the  red  blood-corpuscles  seem 
to  take  part  in  some  way  or  another.  After  excessive  muscular  activity 
the  alkalinity  is  diminished  (Peiper,  Cohnstein),  and  it  is  also  decreased 
after  the  continuous  ingestion  of  acids  (Lassar,  Freudberg^).  Numerous 
investigations  have  been  made  in  regard  to  the  alkalinity  of  the  blood  in 
disease,  but  as  there  is  at  present  no  trustworthy  method  for  estimating- 
the  alkalinity  of  the  blood,  and  as  the  results  are  dependent  upon  the 
indicator  used,  these  investigations,  as  also  the  statements  in  regard  to- 
the  physiological  alkalinity,  require  further  substantiation .^  Attention 
must  also  be  called  to  what  was  stated  (page  191)  in  regard  to  the  determina- 
tion of  the  alkalinity  of  blood-serum — that  determinations  are  made  only 
of  the  titratable  alkali  and  not  of  the  true  alkalinity  caused  by  hydroxy] 
ions. 

The  alkali  of  the  blood  exists  in  part  as  alkaline  salts,  carbonate  and 

*  Zuntz,  Pfliiger's  Arch.,  G6;  Levy-;  Proceed.  Roy.  Soc,  81. 

^  Loewy,  Pfliiger's  Arch.,  58,  which  also  contains  the  references  to  the  literature; 
H.  Strauss,  Zeitschr.  f.  khn.  Med.,  30;  v.  Jaksch,  ibid.,  13;  Peiper,  Virchow's  Arch.. 
116;  Cohnstein,  ibid.,  130,  which  also  cites  the  works  of  Minkowski,  Zuntz,  and  Gej)- 
pert;  Freudberg,  ibid.,  125.  See  also  Weiss,  Zeitschr.  f.  physiol.  Chem.,  38,  Branden- 
burg, Zeitschr   f.  klin.  Med.,  45. 

'  In  regard  to  the  methods  for  the  estimation  of  the  alkalinity  see,  besides  the 
above-mentioned  authors,  v.  Jaksch,  Klin.  Diagnostik;  v  Limbeck,  Wien.  med. 
Blatter,  18;  Wright,  The  Lancet,  1897,  Bifernacki,  Beitriige  zur  Pneumatologie,  etc., 
Zeitschr.  f.  klin.  Med  ,  31  and  32;  Hamburger,  Eine  Methode  zur  Trennung,  etc.,. 
Arch  f.  (Anat.  u.)  Physiol,  1898.  See  also  Maly's  Jahresber.,,  29,  30,  and  31v 
Salaskin  and  Pupkin,  Zeitschr.  f.  physiol.  Chem.,  42,  and  O.  Folin,  ibid.,  43. 


224  THE  BLOOD. 

phosphate^  and  partly  in  combination  with  protein  or  haemoglobin.  The 
first  are  often  spoken  of  as  readily  diffusible  alkalies,  while  the  others  are 
not,  or  are  only  diffusible  with  difficulty  (see  page  187).  The  quantity 
of  the  first  in  human  blood  is  about  one  fifth  of  the  total  alkali  (Branden- 
burg). The  readily  as  well  as  the  difficultly  diffusible  alkali  is  divided 
between  the  blood-corpuscles  and  plasma  and  the  blood-corpuscles  seem 
to  be  richer  in  difficultly  diffusible  alkali  than  the  plasma  or  serum.  This 
division  may  be  changed  by  the  influence  of  even  very  small  amounts  of 
acid,  even  of  carbonic  acid,  and  also,  as  shown  by  Zuntz.  Loew^y  and 
ZuNTZ,  Hamburger,  Limbeck,  and  GiJRBER.^  by  the  influence  of  the  respir- 
atory exchange  of  gas.  The  blood-corpuscles  give  up  a  part  of  the  alkali 
united  with  protein  to  the  serum  by  the  action  of  carbon  dioxide,  hence 
the  senun  becomes  more  alkaline.  The  equilibrium  of  the  osmotic  tension 
in  the  blood-corpuscles  and  in  the  serum  is  hereby  destroyed;  the  blood - 
corpuscles  swell  up  because  they  take  up  water  from  the  serum,  and  this 
then  becomes  more  concentrated  and  richer  in  alkali,  protein,  and  sugar 
Under  the  influence  of  oxygen,  the  corpuscles  take  their  original  form 
again  and  the  above  changes  are  reversed.  The  blood- corpuscles  for  this 
reason  are  less  biconcave  in  their  small  diameter  in  venous  than  in  arterial 
blood  (Hamburger). 

These  conditions  have  been  further  studied  by  v.  Koranyi  and  Bence,^ 
and  they  have  investigated  the  relationship  between  the  changes  of  the 
volume  of  the  blood-corpuscles  and  the  electrical  conductivity,  the  refrac- 
tivity  of  the  serum  and  the  viscosity  of  the  blood.  The  refraction  coefficient 
of  the  serum  is  highest  with  an  increase  in  the  amount  of  carbon  dioxide, 
while  it  is  lowest  when  the  blood  is  rich  in  oxygen  and  poor  in  carbon 
dioxide.  They  consider  this  as  an  action  of  acid,  as  a  similar  rise  is  observed 
after  the  addition  of  acid,  while  after  the  addition  of  alkali  a  fall  in  the 
refraction  coefficient  of  the  serum  takes  place,  and  these  same  changes  can 
be  brought  about  by  CO2  or  by  a  current  of  oxygen.  With  an  increase  in 
the  amount  of  carbon  dioxide,  the  conductivity  of  the  blood  diminishes;  the 
viscosity  is.  on  the  other  hand,  highest  when  the  blood  is  richest  in  carbon 
dioxide.  If  the  CO2  is  driven  off  by  0  the  viscosity  diminishes  to  a  mini- 
mum, and  on  leading  in  more  oxygen  it  rises  again.  The  changes  in  vis 
cosity  of  the  blood  nm  parallel  with  the  volume  changes  of  the  blood-cor- 
puscles, and  changes  in  the  viscosity,  which  can  be  brought  about  by  the 
removal  of  carbon  dioxide,  cause  a  change  in  the  electric  charge  of  the 
blood-corpuscles  (v.  Koranyi  and  Bence). 

'Zuntz  in  Hermann's  Handbuch  der  Physiol.,  4,  Abt.  2;  Loewy  and  Zuntz, 
Pfliiger's  Arch.,  58;  Hamburger,  Arch  f.  (Anat.  u)  Physiol..  1894  and  1898,  and 
Zeitschr.  f.  Biologic,  28  and  35;  v.  Limbeck,  Arch  f.  exp.  Path.  u.  Pharm.,  35;  Gurber, 
Sitzungsber.  d  phys   med.  Gesellsch   zu  Wiirzburg,  1895 

'Pfliiger's  Arch.   110. 


COAGULATION  OF  THE  BLOOD.  225 

The  color  of  the  blood  is  red — light  scarlet-red  in  the  arteries  and  dark 
bluish  red  in  the  veins.  Blood  free  from  oxygen  is  dichroitic,  dark  red 
by  reflected  light  and  green  by  transmitted  light.  The  blood-coloring 
matters  occur  in  the  blood-corpuscles.  For  this  reason  blood  is  opaque  in 
thin  layers.  If  the  hsemoglobin  is  removed  from  the  stroma  and  dissolved 
by  the  blood  liquid  by  an}-  of  the  above-mentioned  means  (see  page  193). 
the  blood  becomes  transparent  and  has  then  a  "lake  color."  "^  Less  light 
is  now  reflected  from  its  interior^  and  this  laky  blood  is  therefore  darker 
in  thicker  layers.  On  the  addition  of  salt  solutions  to  the  blood-corpuscles 
they  shrink,  more  light  is  reflected,  and  the  color  appears  lighter.  A  great 
abimdance  of  red  corpuscles  makes  the  blood  darker,  while  by  diluting 
with  serum  or  by  a  greater  abundance  of  white  corpuscles  the  blood 
becomes  lighter  in  appearance.  The  different  colors  of  arterial  and  of 
venous  blood  depend  on  the  var}-ing  quantities  of  gas  contained  in  these  two 
varieties  of  blood,  or,  better,  on  the  different  amounts  of  oxyhsemoglobin 
and  hsemoglobin  they  contain. 

The  most  striking  property  of  blood  consists  in  its  coagulating  T\ithm  a 
shorter  or  longer  time,  but  as  a  rule  ver}-  shortly  after  leading  the  veins. 
Different  kinds  of  blood  coagulate  viith  var}-ing  rapidity;  in  human  blood 
the  first  marked  sign  of  coagulation  is  seen  in  two  to  three  minutes,  and 
within  seven  to  eight  minutes  the  blood  is  thoroughly  converted  into  a 
gelatinous  mass.  If  the  blood  is  allowed  to  coagulate  slowly,  the  red  cor- 
puscles have  time  to  settle  more  or  less  before  the  coagulation,  and  the  blood- 
clot  then  shows  an  upper  yello\\-ish-gray  or  reddish-gray  layer  consisting 
of  fibrin  enclosing  chiefly  colorless  corpuscles.  This  layer  has  been  called 
crusta  inflammatoria  or  phlogistica,  because  it  has  been  especially  obser\-ed 
in  inflammatory-  processes  and  is  considered  one  of  the  characteristics  of 
them.  This  crusta,  or  "huffy  coat,"  is  not  characteristic  of  any  special 
disease  and  it  occurs  chiefly  when  the  blood  coagulates  slowly  or  when 
the  blood-cor]Duscles  settle  more  quickly  than  usual.  A  buffy  coat  is  often 
observ-ed  in  the  slowly  coagulating  equine  blood.  The  blood  from  the 
capillaries  is  not  supposed  to  have  the  power  of  coagulating. 

Coagulation  is  retarded  by  cooling,  by  diminishing  the  oxygen,  and  by 
increasing  the  amount  of  carbon  dioxide,  which  is  the  reason  that  venous 
blood  and  to  a  much  higher  degree  blood  after  asphyxiation  coagulates 
more  slowly  than  arterial  blood.  The  coagulation  may  be  retarded  or 
prevented  by  the  addition  of  acids,  alkalies,  or  ammonia,  even  in  small 
quantities;  by  concentrated  solutions  of  neutral  alkali  salts  and  alkaline 
earths,  alkali  oxalates  and  fluorides;  also  by  egg-albumin  solutions  of 
sugar  or  gimi,  glycerine,  or  much  water;    also  by  recei%-ing  the  blood  in 


*  R  Du  Bois-Reymond  presents  objections  to  the  general  use  of  the  above  terms 
in  Centralbl.  i  Physiol.,  19,  p.  65. 


226  THE  BLOOD. 

oil.  Coagulation  may  be  prevented  by  the  injection  of  a  proteose  solution 
or  of  an  infusion  of  the  leech  into  the  circulating  blood,  but  this  infusion 
also  acts  in  the  same  way  on  l^lood  just  drawn.  Coagulation  is  also  hindered 
bv  snake  poison  and  toxines  (see  page  171).  The  coagulation  may  be 
facilitated  by  raising  the  temperature;  by  contact  with  foreign  bodies,  to 
which  the  blood  adheres;  by  stirring  or  beating  it;  by  admission  of  air, 
by  diluting  with  very  small  amounts  of  water,  by  the  addition  of  platinum- 
black  or  finely  powdered  carbon;  by  the  addition  of  laky  blood,  which 
does  not  act  by  the  presence  of  dissolved  blood-coloring  matters,  but  by 
the  stromata  of  the  blood-corpuscles;  and  also  by  the  addition  of  the 
leucocytes  from  the  lymphatic  glands,  or  of  a  watery  saline  extract  of  the 
lymphatic  glands,  testicles,  or  thymus  and  various  other  organs  (Dele- 
ZENNE,  Wright,  Arthus,^  and  others). 

An  important  question  to  answer  is  why  the  blood  remains  fluid  in  the 
circulation,  while  it  quickly  coagulates  when  it  leaves  the  circulation.  The 
reason  why  blood  coagulates  on  leaving  the  body  is  therefore  to  be  sought 
for  in  the  influence  which  the  walls  of  the  living  and  uninjured  blood- 
vessels exert  upon  it.  These  \'iews  are  derived  from  the  observations 
of  many  investigators.  From  the  observations  of  Hewson,  Lister,  and 
Fredericq  it  is  known  that  when  a  vein  full  of  blood  is  ligatured  at  the 
two  ends  and  removed  from  the  body,  the  blood  may  remain  fluid  for  a 
long  time.  Brucke  ^  allowed  the  heart  removed  from  a  tortoise  to  beat 
at  0°  C,  and  found  that  the  blood  remained  uncoagiilated  for  some  cLays. 
The  blood  from  another  heart  quickly  coagulated  when  collected  over 
mercury.  In  a  dead  heart,  as  also  in  a  dead  blood-vessel,  the  blood  soon 
coagulates,  and  also  when  the  walls  of  the  vessel  are  changed  by  patho- 
logical processes. 

What  then  is  the  influence  which  the  walls  of  the  vessels  exert  on  the 
liquidity  of  the  circulating  blood?  Freuxd  has  found  that  the  blood 
remains  fluid  when  collected  by  means  of  a  greased  canula  under  oil  or  in  a 
vessel  smeared  with  vaseline.  If  the  blood  collected  in  a  greased  vessel  be 
beaten  with  a  glass  rod  previously  oiled,  it  does  not  coagulate,  but  it 
quickly  coagulates  on  beating  it  with  an  unoiled  glass  rod  or  when  it  is 
poured  into  a  vessel  not  greased.  The  non-coagulability  of  blood  collected 
under  oil  was  confirmed  later  by  Haycraft  and  Carlier.  Freuxd  found 
on  further  investigation  that  the  evaporation  of  the  upper  layers  of 
blood  or  their  contamination  with  small  quantities  of  dust  causes  a  coami- 

'  Delezenne,  .\rch.  de  Physiol.  (5),  8;  Wright,  Journ.  of  Physiol.,  28;  Arthus,  Journ. 
de  Physiol,  et  Pathol.,  4. 

■*  Hewson's  works,  edited  by  Gulliver,  London,  1876,  cited  from  Gamgee,  Text- 
book of  Physiol.  Chem  ,  1,  1880;  Lister,  cited  from  Gamgee,  ibid.;  Fredericq, 
Recherclies  .sur  la  con.stitution  du  plasma  .sanguin,  Gand,  1878,  Briicke,  Virchov\'s 
Arch.,  12. 


COAGULATION   OF  THE   BLOOD.  227 

lation  even  in  a  vessel  treated  with  vaseline.  According  to  Freuxd  ^  it 
is  tliis  adhesion  between  the  blood  or,  as  the  blood  shows  an  adhesion  to 
the  normal  vessel  walls  (Benno  Lewy),  between  its  form-elements  and  a 
foreign  substance — and  the  diseased  walls  of  the  vessel  also  act  as  such — 
that  gives  the  impulse  towards  coagulation,  while  the  lack  of  adhesion 
prevents  the  blood  from  coagvilating.  Bordet  and  Gengou  ~  have  also 
shown  that  the  plasma  obtamed  by  centrifuging  blood  collected  in  a  paraf- 
fined vessel,  and  perfectly  free  from  form-elements,  can  be  kept  without 
coagulating  m  a  paraffined  vessel,  and  that  it  does  coagulate  on  being  trans- 
ferred to  an  unparaffined  vessel.  The  adhesion  of  the  plasma  to  a  foreign 
body  may  also,  in  the  absence  of  form-elements,  give  the  impulse  to  coagu- 
lation. That  this  adhesion  of  the  form-elements  is  of  great  importance 
cannot  be  denied  and  is  also  generally  accepted.  By  this  adhesion  the 
form-elements  undergo  certain  changes  which  seem  to  stand  in  a  certain 
relationship  to  the  coagulation  of  the  blood. 

The  views  in  regard  to  these  changes  are,  unfortunately,  very  contra- 
dictor}\  Accordmg  to  Alex.  SchxMidt  ^  and  the  Dorpat  school  an 
abundant  destruction  of  the  leucocytes,  especially  polynuclear  leucocytes, 
takes  place  in  coagulation  and  important  constituents  for  the  coagulation 
of  the  fibrin  pass  into  the  plasma.  A  direct  relationship  between  the  de- 
struction of  leucocytes  and  coagulation  is  denied  by  many  investigators,, 
while  according  to  other  experimenters  the  essential  is  not  a  destruction 
of  the  leucocytes,  but  an  elimination  of  constituents  from  the  cells  into 
the  plasma.  This  process  is  called  plasmoschisis  by  LowaT.'*  The  passage 
of  cell  constituents  into  the  plasma  before  coagulation  must  not  neces- 
sarily be  considered  as  a  phenomenon  of  death,  as  it  may  just  as  well  be 
a  secretory  process  (Arthus,  [NIorawitz,  Dastre^).  Great  importance 
has  also  been  ascribed  to  the  blood-platelets  in  coagulation,  as  certain 
investigators  (Bizzozero,  Lilienfeld,  Schwalbe,  ^Morawitz,  Burker) 
have  found  that  they  cause  or  accelerate  coagulation,  while  others  (Petroxe) 
on  the  contrar}^  fird  a  retarding  action.^ 

'  Freund,  Wien.  med.  Jahrb.,  1886;  Haycraft  and  Carlier,  Journ.  of  Anat.  and 
Physiol.,  22;  Benno  Lewy,  Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Suppl. 

^  Annal.  de  I'lnstitute  Pasteur,  1". 

^  Pflrger's  Arch.,  11.  Tlip  works  of  Alex.  Schmidt  are  found  in  Arch.  f.  Anat.. 
und  Physiol.,  1861,  1862;  Pfl.iger's  Arch.,  6,  9,  11,  13.  See  especially  Ale.x.  Schmidt, 
Zur  Blutlehre  (Leipzig,  1892),  wliich  also  gives  the  work  of  his  pupils,  and  Weitere. 
Beitrage  zur  Blutlehre,  1895. 

^  Wien.  Sitzungsber.,  89  and  90,  and  Prager  med.  Wochenschr.,  1889.  referred, 
to  m  Centralbl.  f.  d.  med.  Wissenscli.,  28,  265. 

^  Morawitz,  Hofmeister's  Beitrage,  5;  Arthus,  Compt.  rend.  .'oc.  biolog.,  55;  Dastre,. 
ibid.,  55. 

*•  See  foot-note  2,  p.  222.  Also  Schwalbe,  Unters.  z.  Blutgerinnvmg,  etc.,  Braun- 
schweig, 1900;  Morawitz,  Deutsch.  Arch.  f.  klin.  Med.,  "9,  and  Hofmeister's  Beitrage^ 
4  and  5,   B  .rker,  Pfl'ger's  Arch.,  102,   Petrone,  Maly's  Jahresber.,  31,  p.  170. 


228  THE  BLOOD. 

WooLDRiDGE  '  takes  a  very  peculiar  position  in  regard  to  this  question: 
he  considers  the  form-elements  as  only  of  secondary  importance  in  coagulation. 
As  he  has  found,  a  peptone-plasma  which  has  been  freed  from  all  form-con- 
stituents by  means  of  centrifugal  force  yields  abundant  fibrin  when  it  is  not 
separated  from  a  substance  which  precipitates  on  cooling.  This  substance, 
which  WooLDRiDGE  has  called  A-fibrinogen,  seems  to  all  appearances  to  be  a 
nucleoproteid,  which,  according  to  the  unanimous  view  of  several  investigators, 
originates  from  the  form-elements  of  the  blood,  either  the  blood-plates  or  the 
leucocytes,  and  the  generally  accepted  view  as  to  the  great  importance  of  the 
form-elements  in  the  coagulation  of  the  blood  is  not  really  contrary  to  Wool- 
dridge's  experiments. 

The  views  are  greatly  di^'ided  in  regard  to  those  bodies  w^hich  are  elim- 
inated from  the  form-elements  of  the  blood  before  and  during  coagulation. 

According  to  Alex.  Schmidt  the  leucocytes,  like  all  cells,  contain  two 
chief  groups  of  constituents,  one  of  which  accelerates  coagulation,  while  the 
other  retards  or  hinders  it.  The  first  may  be  extracted  from  the  cells  by 
alcohol,  while  the  other  cannot  be  extracted.  Blood-plasma  contains  only 
traces  of  thrombin,  according  to  Schmidt,  but  does  contain  its  antecedent, 
prothrombin.  The  bodies  which  accelerate  coagulation  are  neither  thrombin 
nor  prothrombin,  but  they  act  in  this  wise  in  that  they  split  off  thrombin 
from  the  prothrombin.  On  this  account  they  are  called  zymoplastic  sub- 
stances by  Alex.  Schmidt.  The  nature  of  these  bodies  is  unknown,  and 
Schmidt  has  given  no  notice  of  their  beha\dor  with  the  lime  salts,  which 
have  been  found  to  have  zymoplastic  activity  by  other  investigators. 

The  constituents  of  the  cells  which  hinder  coagulation  and  which  are 
insoluble  in  alcohol-ether  are  compound  proteids  and  have  been  called 
cytoglobin  and  preglohulin  by  Schmidt.  The  retarding  action  of  these 
bodies  may  be  suppressed  by  the  addition  of  zymoplastic  substances,  and 
the  yield  of  fibrin  on  coagulation  in  this  case  is  much  greater  than  in  the 
absence  of  the  compound  proteid  retarding  coagulation.  This  last  supplies 
the  material  from  which  the  fibrin  is  produced.  The  process  is,  according 
to  Schmidt,  as  follows:  The  preglobulin  first  splits,  yielding  serglobulin, 
then  from  this  the  fibrinogen  is  derived,  and  from  this  latter  the  fibrin  is 
produced.  The  object  of  the  thrombin  is  twofold.  The  thrombin  first 
splits  the  fibrinogen  from  the  paraglobulin  and  then  converts  the  fibrinogen 
into  fibrin.  The  assumption  that  fibrinogen  can  be  split  from  paraglobulin 
has  not  sufficient  foundation  and  is  even  improbable. 

According  to  Schmidt  the  retarding  action  of  the  cells  is  prominent 
during  life,  while  the  accelerating  action  is  especially  pronounced  outside 
of  the  body  or  by  coming  in  contact  with  foreign  bodies.  The  parenchy- 
mous  masses  of  the  organs  and  tissues,  through  which  the  blood  flows  in 
the  capillaries,  are  those  cell-masses  which  serve  to  keep  the  blood  fluid 
during  life. 

^  Die  Gerinnung  des  Blutes  (published  by  M.  v.  Frey,  Leipzig,  1891). 


COAGULATION  OF  THE  BLOOD.  229 

LiLiENFELD  has  given  further  proof  as  to  the  occurrence  in  the  form- 
elements  of  the  blood  of  bodies  which  accelerate  or  retard  the  coagulation. 
According  to  this  author  the  nature  of  these  bodies  is  ver}-  markedly  differ- 
ent from  Schmidt's  idea.  While,  according  to  ScH^noT,  the  coagulation 
accelerators  are  bodies  soluble  in  alcohol,  and  the  compound  proteids 
exhausted  with  alcohol  act  only  retardingly  on  coagulation,  Liliexfeld 
states  that  the  substance  which  acts  acceleratingly  and  retardingly  on 
coagulation  consists  of  a  nucleoproteid,  namely,  nucleohistone.  Nucleo- 
histone  readily  splits  into  leuconuclein  and  histone,  the  first  of  which  acts 
as  a  coagulation-excitant,  while  the  other,  mtroduced  into  the  blood-vascular 
system,  either  intravascular  or  extravascular,  robs  the  blood  of  its  property 
of  coagulating.  Introduced  into  the  circulator}'  system  the  nucleohistone 
splits  into  its  two  components.  It  therefore  causes  extensive  coagulation 
on  one  side  and  makes  the  remainder  of  the  blood  uncoagulable  on  the 
other.  This  theory  as  well  as  that  of  Schmidt  is  not  based  upon  sufficiently 
positive  facts. 

Beucke  showed  long  ago  that  fibrin  left  an  ash  containing  calcium 
phosphate.  The  fact  that  calcium  salts  may  facilitate  or  even  cause  a 
coagulation  in  liquids  poor  in  ferment  has  been  known  for  several  years 
through  the  researches  of  Hammarstex,  Greex,  Rixger  and  Saixsbury. 
The  necessity  of  the  lime  salts  for  the  coagulation  of  blood  and  plasma  was 
first  shown  positively  by  the  important  investigations  of  Arthus  and 
Pages.  Recent  investigations  of  Sabbataxi  ^  have  also  shown  the  impor- 
tance of  calcium  salts  or  the  free  calcium  ions  for  coagulation  without 
explaining  the  mode  of  their  action. 

According  to  the  generally  accepted  view  of  Arthus  and  Pages  the  soluble 
lime  salts  precipitable  by  oxalate  are  necessary  requisites  for  the  fermentive 
transformation  of  fibrinogen,  because  thrombin  remains  inactive  in  the  absence 
of  soluble  lime  salts.  This  view  is  untenable,  as  shown  by  the  researches  of 
Alex.  Schmidt,  Pekelharixg,  and  Hammarstex.'  Thrombin  acts  as  well  in 
the  absence  as  in  the  presence  of  precipitable  lime  salts. 

Liliexfeld's  theory  that  the  leuconuclein  splits  off  a  protein  substance, 
thrombosinjrom  the  fibrinogen,  and  that  this  thrombosin  forms  an  insoluble  com- 
pound with  the  lime  present,  producing  thrombosin  lime  (fibrin),  which  separates, 
is  incorrect  according  to  Hammarstex,  Sch'afer,  and  Cramer.^  Liliexfeld's 
thrombosin  is  nothing  but  fibrinogen  which  is  precipitated  by  a  lime  salt  from  a 
salt-poor  or  salt-free  solution. 

'  Hammarsten,  Nova  Acta  reg.  Sec.  Scient.  Upsal.  (3).  10,  1879;  Green,  Joum.  of 
Physiol.,  8;  Ringer  and  Sainsbury,  ibid.,  11  and  12,  Arthus  et  Pages  and  Arthus, 
see  foot-note  4,  p.  171;  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22;  Sabbatani, 
cited,  Centralbl.  f.  Physiol.,  16,  665. 

^  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22,  where  the  other  investigators  are 
cited. 

'Hammarsten,  1.  c;  Schafer,  Journ.  of  Physiol.,  17;  Cramer,  Zeitschr.  f.  physiol. 
Chem.,  23. 


230  '      THE  BLOOD. 

According  to  Pekelharing  ^  thrombin  is  the  lime  compound  of  pro- 
thrombin, and  the  process  of  coagulation  consists,  according  to  him,  in 
the  thrombin  transferring  the  lime  to  the  fibrinogen,  which  is  hereby  con- 
verted into  an  insoluble  lime  compound,  fibrin.  Of  the  objections  to  this 
theory  can  be  mentioned,  among  others,  the  fact  that  fibrin  has  been 
obtained  not  absolutely  free  from  lime,  but  still  so  poor  in  lime  (Hammar- 
STEN  2)  that  if  the  lime  belongs  to  the  fibrin,  its  molecule  must  be  more 
than  ten  times  greater  than  the  haemoglobin  molecule,  which  is  not  prob- 
able. These  as  well  as  many  other  observations  indicate  that  the  lime 
is  carried  down  by  the  fibrinogen  only  as  a  contamination. 

If,  as  it  seems,  the  lime  is  not  of  importance  in  the  transformation  of 
fibrinogen  into  fibrin  in  the  presence  of  thrombin,  still  this  does  not  con- 
tradict the  above-mentioned  observations  of  Arthus  and  Pages  that  the 
lime  salts  are  necessary  for  the  coagulation  of  blood  and  plasma.  It  is  very 
probable  that  the  lime  salts,  as  admitted  by  Pekelharing,  are  a  necessary 
requisite  for  the  transformation  of  prothrombin  into  thrombin. 

If  we  attempt  to  summarize  the  more  or  less  contradictory  investiga- 
tions and  views  as  given  m  the  preceding  pages,  we  can  consider  the  follow- 
ing facts  as  conclusive:  In  the  first  place,  two  bodies,  the  fibrinogen  and 
the  thrombin,  are  necessary  for  the  coagulation.  Tlie  fibrinogen  exists 
preformed  in  the  plasma.  The  thrombin,  on  the  contrar}^,  does  not  occur 
in  living  blood,  at  least  not  in  appreciable  amounts  as  such,  but  is  formed 
from  another  substance,  the  prothrombin.  The  presence  of  calcium  salts 
is  necessary  for  the  formation  of  this  thrombin,  while  the  calcium  salts 
are  not  necessan,^  for  the  enzymotic  transformation  of  fibrinogen  into  fibrin. 
Besides  the  calcium  salts  also  other  substances,  the  zymoplastic  active 
substances,  are  active  in  the  formation  of  thrombm  from  its  mother- 
substance,  and  these  zymoplastic  substances  stand  in  some  relation  to 
the  form-elements  of  the  blood. 

The  formation  of  thrombin  and  the  relationship  of  the  form-elements 
therewith  are  still  unexplained  or  disputed  questions. 

It  is  a  question  whether  the  mother-substance  of  thrombin  exists  in 
the  plasma  of  the  circulating  blood  or  whether  it  is  a  body  eliminated  from 
the  form-elements  before  coagulation.  We  have  two  contradictory  views 
on  this  question,  namely,  those  of  Alex.  Schmidt  and  of  Pekelharing. 
According  to  Schmidt  prothrombin  occurs  preformed  in  the  circulating 
plasma,  and  it  is  transformed  into  thrombin  by  the  zymoplastic  substances 
which  pass  out  from  the  form-elements.  Pekelharing,  on  the  contrary, 
holds  the  view  that  the  plasma  does  not  contain  appreciable  amounts 
of  prothrombin.     This  body,  according  to  him,  passes  before  coagulation 

'  See  foot-note  6,  p.  175,  and  especially  Virchow's  Festschrift,  1,  1891. 
*  Zeitschr.  f.  physiol.  Chem.,  2S. 


COAUULATIOX  OF   THE  BLOOD.  231 

from  the  form-elements  into  the  plasma,  and  is  there  converted  into  thrombin 
b}'  the  calcium  salts.  The  observation  that  uncoagulated  leech-plasma 
does  not  coagulate  on  the  addition  of  calcium  salts,  while  it  does  coagulate 
on  the  addition  of  prothrombin  solutions,  seems  to  support  this  Anew;  still 
it  is  not  quite  conclusive.  Leech-extract  contains  a  body,  hirudin,  which, 
according  to  ^Morawitz,  is  an  antibody  towards  thrombin  and  quanti- 
tatively neutralizes  it.  On  the  addition  of  prothrombin,  new  thrombin 
may  be  formed,  which  may  act  if  the  hirudin  is  not  present  in  too  great  an 
excess. 

The  behavior  of  sodium-fluoride  plasma  shows  more  conclusively  the 
absence  of  prothrombin  m  the  circulating  plasma.  Such  plasma,  according 
to  Arthus,  contains  no  prothrombin,  a  statement  which  has  been  partly 
substantiated  by  ^Iorawitz,  who  finds  that  fluoride-plasma  contains  more 
or  less  prothrombin,  dependent  upon  the  greater  or  less  change  the  blood 
undergoes  before  it  flows  into  the  sodium-fluoride  solution.  One  can 
obtain,  according  to  Morawitz,  at  least  sometimes,  a  fluoride-plasma 
which  contains  no  prothrombin.  The  observations  of  Fuld  and  of  Schit- 
TENHELM  and  BoDONG  contradict  the  statement  that  fluoride-plasma 
contains  prothrombin.  As  Bordet  and  Gen'gou  ^  have  sho\\7i  that  pro- 
thrombin can  be  carried  down  by  the  precipitate  produced  in  fluoride- 
plasma,  it  seems  as  if  the  observations  of  Arthus  and  ^Morawitz  on  this 
point  are  not  conclusive,  and  it  is  probable  that  all  plasma  contams  pro- 
thrombin. The  absence  of  prothrombin,  as  observed  by  Arthus,  in 
peritoneal  transudates  in  the  horse,  can  hardly  be  considered  as  sufficient 
evidence  as  to  the  occurrence  of  this  body  in  blood-plasma. 

The  unsettled  condition  of  the  question  of  the  zymoplastic  substances 
has  recently  been  somewhat  enlightened,  and  the  starting-point  in  these 
new  investigations  is  the  accelerating  action  upon  coagulation,  of  different 
tissue  extracts,  an  action  which  has  been  known  for  a  long  time  and  was 
especially  studied  by  Delezenne  on  the  plasma  from  bird's  blood.  The  active 
constituent  of  these  tissue  extracts  is  called  thromhokinase  by  ^Iorawitz, 
and,  according  to  him,  this  thromhokinase  is  necessary-,  besides  Ume-salts,for 
the  transformation  of  prothrombin  (thrombogen  according  to  Morawitz). 
The  production  of  thromhokinase  is,  according  to  ^Iorawitz,  a  general 
property  of  the  protoplasm  and  occurs  also  in  the  leucocytes.  The  throm- 
hokinase of  drawn  blood  originates  in  birds  and  m  part  in  mammals  from 
the  leucocytes.  In  mammalian  blood  the  blood-plates  are  the  essential 
source.     For   the   formation   of  thrombin   three  different  substances   are 

'  Arthus,  Journ.  de  Physiol,  et  Pathol.,  3  and  4,  and  Compt.  rend.  soc.  biol.,  56. 
The  works  of  Morawitz  may  be  found  in '  Hofmeister's  Beitrage,  4  and  5,  Deutsch. 
Arch.  f.  klin.  Med.,"9and  80,  and  Centralbl.  f.  Physiol.,  17,  p.  529;  with  Spiro,  Hof- 
meister's  Beitrage,  5;  Schittenhelm  and  Bodong,  Arch.  f.  exp.  Path.  u.  Pharm.,  51; 
Bordet  and  Gengou,  Annal.  Institut  Pasteur,  IS. 


232  THE  BLOOD. 

necessan-  according  to  Morawitz,  namely,  thrombogen,  thrombokinase, 
and  lime-salts.  Thrombogen  is,  according  to  Morawitz,  not  quite 
identical  with  the  prothrombin  (other  investigators),  which  he  calls  a-pro- 
thrombin,  but  is  a  mother-substance  of  the  same.  The  process  of  thrombin 
formation  can  be  given  as  follows:  the  kinase  first  transforms  the  throm- 
bogen into  a-prothrombin,  which  latter  then  is  converted  into  thrombin 
(a)  by  the  lime-salts. 

Thrombokinase  is  precipitated  by  alcohol  and  is  not  resistant  towards 
heat.  It  therefore  cannot  be  identical  with  Schmidt's  zymoplastic  sub- 
stances, and  this  point  requires  further  elucidation.  The  thrombokinase 
also  does  not  occur  to  any  appreciable  extent  in  the  circulating  blood. 
The  accelerating  action  upon  coagulation  of  tissues  or  parts  of  tissues  de- 
pends, as  above  stated,  upon  their  content  of  kinase;  but  it  also  in  part 
depends  upon  the  fact  that  the  tissue  fluids  excite  the  secretory  acti\'ity 
of  the  form-elements. 

FuLD^  has  arrived  at  about  the  same  results  independently  of  Mora- 
wiTz,  but  he  has  selected  other  names.  The  three  substances  thrombogen, 
kinase,  and  thrombin  are  called  by  him  plasmozym,  cytozym,  and  holozym. 
The  chief  reason  why  circulating  blood  remains  fluid  is,  according  to 
FuLD.  because  the  cytozj-m  is  only  slowly  formed  therein  and  the  ferment 
(holozym)  produced  thereby  is  in  an  inactive  form.  Another  reason  is  that 
the  blood  contains  an  antibody  for  the  fibrin  ferment.  The  assumption 
of  the  presence  of  an  antibody,  generally  antithrombin,  in  the  circulating 
blood,  which  retards  coagulation,  does  not  only  seem  to  be  probable,  but 
recently  Pugliese  -  has  isolated  an  antithrombin  from  blood  and  tissues. 

A  serum  poor  in  ferment  and  ha^Tng  a  weak  action  can  be  reactivated  by  the 
addition  of  acid  or  alkali  (Alex.  Schmidt,  Morawitz),  and  in  this  action,  accord- 
ing to  MoRA^v^TZ,  a  thrombin  (,3)  is  produced  which  is  somewhat  different  from 
a-thrombin.  The  f3-thrombin  is  produced  from  a  special  ^-prothrombin  which  never 
occurs  in  the  plasma,  but  only  in  the  serum.  Fuld  explains  this  by  the  state- 
ment that  the  a-thrombin  is  changed  in  the  serum  into  metazym  (/3-prothrombin), 
which  is  then  transformed  by  the  alkali  or  acid  into  neozym  (  =  ^-thrombin). 

L.  LoEB,3  who  has  also  conducted  extensive  investigations  on  the  coagu- 
lation of  the  blood,  ascribes,  like  other  investigators,  a  great  importance 
to  the  bodies  existing  in  the  tissue,  which  accelerate  coagulation,  and  to 
which  he  gives  the  name  tissue  coagulins.  These  coagulins  are  indeed  not 
identical  with  the  coagulins  of  the  blood-clot  or  the  blood-serum,  but,  like 
these,  act  directly  upon  fibrinogen.  Under  favorable  conditions  the  com- 
bined action  of  blood  and  tissue  coa2:ulins  mav  be  greater  than  the  sum 


'  Centralbl.  f.  Physiol.,  17.     See  also  Fuld  and  Spire,  Hofmeister's  Beitrage,  5. 
'  Journ.  de  Physiol.,  7. 

^  The  work  of  Loeb  may  be  foimd  in  Medical  News,  New  York,  1903.     Virchow's 
Arch.,  176,  and  Hofmeister's  Beitrage,  5. 


INTRAVASCUL-YR  COAGULATION.  233 

of  the  indi\'idual  actions.  He  explains  this  by  stating  that  an  activation 
takes  place  by  means  of  a  kinase;  still,  though  this  is  possible,  he  has  not 
proved  it. 

The  coagulins  of  the  blood  are,  according  to  Loeb,  different  from  the  tissue 
coaguiins.  The  first  show  no  specific  action,  i.e.,  not  between  invertebrates  and 
vertebrates.  The  tissue  coagulins,  on  the  contrary,  have  by  their  action  upon  the 
blood  a  certain  specificity,  at  least  in  animals  widely  separated  from  one  another. 

Based  upon  recent  investigations,  a  short  summary  of  the  coagulation 
of  the  blood  would  be  as  follows:  In  the  circulating  blood-plasma  there 
occur  fibrinogen,  lime  salts,  and  probably  also  prothrombin.  On  aecoimt 
of  the  continued  destruction  of  small  amoimts  of  form-elements,  probably 
small  quantities  of  thrombin  are  formed,  which  is  destroyed  or  made 
inactive  by  the  simultaneous  presence  of  antithrombin.  The  reason  why 
the  blood  remains  fluid  in  life  lies  in  the  lack  of  thrombin.  Under  the 
influence  of  foreign  bodies  or  of  chemical  irritants  within  or  outside  of 
the  body  the  form-elements  of  the  blood  are  incited  to  an  increased  secretory 
acti%-ity.  and  from  them  (perhaps  also  from  the  leucocytes  in  o\'ipara  or 
from  the  leucocytes  but  chiefly  from  the  blood-plates  in  mammalia)  an 
abundance  of  kinase  passes  into  the  plasma.  By  this  (as  well  as  by  the 
action  of  tissue  fluids  outside  of  the  body)  the  thrombogen  is  transformed 
into  a-prothrombin,  which  is  changed  by  the  lime  salts  into  thrombin 
(a-thrombin).    The  latter  transforms  the  fibrinogen  into  fibrin. 

The  bodies  accelerating  coagulation,  like  the  tissue  extracts  and  the 
lime  salts,  act  upon  the  formation  of  thrombin.  The  mode  of  action  of 
gelatine,  if  it  has  any  accelerating  action  at  all,  is  not  knoA^-n.  The  bodies 
retarding  coagulation  may  in  certain  cases  act  directly  upon  the  blood,, 
either,  like  the  neutral  salts,  retarding  the  development  of  the  thrombin, 
or.  like  the  oxalate  or  fluoride,  preventing  the  same;  or  like  the  hirudin,^ 
which,  as  an  antithrombm,  makes  the  thrombin  inactive;  or  like  the  cobra- 
poison,  which  acts  like  an  antikinase.  In  other  cases  they  may  have  an 
mdirect  action,  for  they  may,  like  the  proteoses  and  others,  cause  the 
body  to  produce  special  bodies  which  stand  in  close  relation  to  intra- 
vascular coagulation. 

Intravascular  Coagulation.  It  has  been  sho^-n  by  Alex.  Schmidt  and 
his  students,  as  also  by  Wooldridge,  Wright,-  and  others,  that  an  intra- 
vascular coagulation  may  be  brought  about  by  the  intravenous  injection 
into  the  circulating  blood  of  a  large  quantity  of  a  thrombin  solution,  as 
also  by  the  injection  of  leucocytes  or  tissue  fibrinogen  (impure  nucleopro- 

*  The  action  of  hirudin  is  somewhat  doubtful.     See  Schittenhelm  and  Bodong,  I.e. 

'A  Study  of  the  Intravascular  Coagulation,  etc..  Proceed,  of  the  Roy.  Irish  Acad. 
(3),  2.  See  also  Wright,  Lecture  on  Tissue  or  Cell  Fibrinogen,  The  Lancet,  1892; 
and  Wooldridge's  Method  of  Producing  Inunimity,  etc.,  Brit.  Med.  Joiu-nal,  Sept.,  1S9L 


234  THE   BLOOD. 

teid).  Intravascular  coagulation  may  be  brought  about  also  under  other 
conditions,  such  as  after  the  injection  of  snake-poison  (iNIartin  ^  and  others) 
or  certain  of  the  proteid-like  colloid  substances,  sjTithetically  prepared 
according  to  Grimaux's  method  (Halliburton  and  Pickering  2).  If  too 
little  of  the  above-mentioned  bodies  be  injected,  then  we  observe  only  a 
marked  retarding  tendency  in  the  coagulation  of  the  blood.  According  to 
WooLDRiDGE  it  can  generally  be  maintained  that  after  a  short  stage  of 
accelerated  coagulability,  which  may  lead  to  a  total  or  partial  intravascular 
coagulation,  a  second  stage  of  a  diminished  or  even  arrested  coagulability 
of  the  blood  follows.  The  first  stage  is  designated  (Wooldridge)  as  the 
positive  and  the  other  as  the  negative  phase  of  coagulation.  These  state- 
ments have  been  confirmed  by  several  investigators. 

There  is  no  doubt  that  the  positive  phase  is  brought  about  by  an  abun- 
dant introduction  of  thrombin,  or  by  a  rapid  and  abundant  formation  of 
the  same.  The  explanation  of  the  production  of  the  negative  phase,  which 
<;an  easily  be  brought  about  by  pepsin  proteoses,  by  various  bodies  such  as 
extracts  of  crabs'  muscles  and  other  organs,  eel-serum,  enzymes,  bacterial 
toxines,  snake-poisons,  etc.,  has  been  attempted  in  different  ways.  The 
best  studied  is  the  action  of  proteoses,  but  no  conclusive  results  have 
been  obtained  thus  far.  The  assertion  of  Pick  and  Spiro  that  the  action 
of  the  proteoses  does  not  depend  upon  the  proteoses  themselves,  but  upon 
a  contaminating  substance,  the  protozym,  has  been  shown  to  be  incorrect 
by  Underbill.  The  bodies  retarding  coagulation  obtained  by  Conradi^ 
m  autolysis,  which  are  probably  antithrombins,  seem  to  act  in  a  different 
way  from  the  proteoses,  and  cannot  for  the  present  be  made  use  of  in  ex- 
plaining this  question. 

There  are  a  large  number  of  researches  on  the  action  of  proteoses  and 
oi  other  retarding  substances  by  different  investigators,  such  as  Grosjean, 
Ledoux,  Contejean,  Dastre,  Floresco,  Athanasiu,  Carwallo,  Gley, 
Pachon,  Spiro  and  Ellinger,  Fuld  and  Spiro,  Morawitz  and  Nolf,  but 
those  of  Delezenne  *  are  of  the  greatest  importance.  We  can  say  with 
certainty  that  the  action  is  indirect  and  that  the  liver,  and  perhaps  also  the 
leucocytes  and  vessel  walls  (Nolf),  are   important  for   the  process.     The 

'  Journ.  of  Physiol.,  15. 

'  Ibid.,  18. 

'  Pick  and  Spiro,  Zeitschr.  f.  physiol.  Chem.,31;  Conradi,  Hofmeister's  Beitrage,  1. 
"See  also  Underbill,  Amer.  Journ.  of  Physiol.,  9. 

^  Grosjean,  Travaux  du  laboratoire  de  L,  Fredericq,  4,  Liege,  1892;  Ledoux,  ibid., 
5, 1896;  Nolt,  Bull.  I'Acad.  roy.  de  Belgique,  1902  and  1905,  and  Biochem.  Centralbl.,3; 
Spiro  and  Ellinger,  Zeitschr.  f.  physiol.  Chem.,  23;  Fuld  and  Spiro,  1.  c;  Morawitz, 
1.  c.  The  works  of  the  above-mentioned  French  investigators  can  be  found  in  Compt. 
Tend.  soc.  biol.,  46,  47,  48,  50,  and  51,  and  Arch.  d.  Physiol.  (6),  7,  8,  9,  and  10;  see 
also  especially  Delezenne,  Arch.  d.  Physiol.  (6),  10;  Compt.  rend,  soc  biol.,  51,  and 
Compt.  rend.,  130. 


QUANTITATIVE    COMPOSITION    OF    THE   BLOOD.  23o 

reasons  for  the  non-coagulability  of  "peptone  blood"  are  of  two  kinds:  first, 
this  blood  contains  an  antithrombin,  and,  secondly,  the  thrombin  for  inex- 
plicable reasons  is  absent,  although  such  blood  seems  to  contain  thrombogen 
as  well  as  kinase.  The  reason  for  the  insufficient  formation  of  thrombin 
is  unknown,  and  only  a  few  observations  have  been  collected  on  the  forma- 
tion of  antithrombin.  According  to  Xolf.  the  peptone  (more  correctly 
the  proteoses)  causes  an  alteration  in  the  leucocytes  and  the  walls  of  the 
vessels,  and  a  substance  is  secreted  which  brings  about  in  the  liver  the 
formation  of  antithrombin.  According  to  Delezexxe,  the  proteoses  bring 
about  a  destruction  of  leucocytes,  and  thereby  a  substance  accelerating 
coagulation  and  another  ha^-ing  a  retarding  action  are  set  free.  The  first 
is  destroyed  by  the  liver,  and  hence  the  action  of  the  retarding  substance 
(the  antithrombin)  is  obtained.  The  only  thing  that  is  positivel}'  proven 
is  the  part  taken  by  the  liver  in  this  retardation  of  coagulation,  as  sho^-n 
by  Gley  and  Pachon;  the  non-appearance  of  the  thrombin  formation  is 
not  explained  by  the  above  theories. 

The  coagulation  of  the  blood  of  lower  animals  may  be  of  two  kinds,  according 
to  L.  LoEB.^  A  partial  agglutination  of  the  blood-cells  may  take  place,  and  this 
kind  of  coagulation  is  the  only  kind  in  certain  animals;  but  a  true  coagulation 
of  fibrinogen  may  also  take  place.  This  latter  coagulation  is  essentially  the  same 
as  occurs  in  vertebrates,  and  here  also  an  action  of  kinase  (coagulin)  upon  throm- 
bogen takes  place. 

The  non-coagulability  of  cadaver  blood  depends  usually,  according  to  Mora- 
WITZ,^  upon  the  fact  that  it  contains  no  fibrinogen,  due  to  a  fibrinolysis. 

The  gases  of  the  blood  will  be  treated  of  m  Chapter  XVII  (on  respira- 
tion) . 

IV.    The  Quantitative  Composition  of  the  Blood. 

The  quantitative  analyses  of  the  blood  are  of  little  value.  We  must 
ascertain  on  one  side  the  relationship  of  the  plasma  and  blood-corpuscles  to 
each  other,  and  on  the  other  side  the  constitution  of  each  of  these  two 
chief  constituents.  The  difficulties  which  stand  in  the  way  of  such  a  task, 
especially  in  regard  to  the  li^■ing,  non-coagidated  blood,  have  not  been 
removed.  Since  the  constitution  of  the  blood  may  differ  not  only  in  differ- 
ent vascular  regions,  but  also  in  the  same  region  under  different  circum- 
stances, which  renders  also  a  number  of  blood  analyses  necessar}',  it  can 
hardly  appear  remarkable  that  our  knowledge  of  the  constitution  of  the 
blood  is  still  relatively  limited. 

The  relative  volume  of  blood-corpuscles  and  senmi  in  defibrinated  blood 
may  be  determined .   according   to  L.   and  M.   Bleibtreu.^  by   various 

'  Hofmeister's  Beitrage,  5  and  6,  and  Virchow's  Arch.,  176.     See  also  Ducceschi, 
Hofmeister's  Beitrage,  3. 
'  Hofmeister's  Beitrage,  S. 
'  Pfliiger's  Arch.,  51.  55,  and  60. 


236  THE  BLOOD. 

methods  if  the  defibrinated  blood  is  mixed  with  different  proportions  of 
isotonic  NaCl  solution  (1  vol.  of  the  blood  to  at  least  1  vol.  salt  solution), 
the  blood-corpuscles  allowed  to  settle  to  the  bottom,  which  may  be  facili- 
tated by  centrifugal  force,  and  the  clear  supernatant  mixture  of  serum 
and  salt  solution  siphoned  off.     The  methods  are  as  follows: 

1.  Determine  the  quantity  of  nitrogen  in  at  least  two  different  portions  of 
the  mixture  of  serum  and  salt  solution  by  means  of  Kjeldahl's  method  and 
calculate  the  quantity  of  protein  corresponding  thereto  by  multiplying  with 
6.25:  and  the  relative  volume  of  blood  x,  and  also  the  volume  of  the  structural 
elements  (1  —x),  are  found  by  the  following  equation: 

/    -,    £2    _  fi 

In  this  equation  (for  mixtures  1  and  2)  6,  or  h^  represents  the  volume  of  blood 
in  the  mixture,  s,  or  8^  the  volume  of  salt  solution,  and  e-^  or  e^  the  quantity  of 
protein  in  a  certain  volume  of  each  mixture. 

2.  Determine  the  specific  gravity  of  the  blood-serum,  of  the  salt  solutions,  and 
of  at  least  one  of  the  mixtures  of  serum  and  salt  solution  by  means  of  a  pycnom- 
eter.    The  relative  volume  of  serum  x  is  found  in  this  by  the  following  equation: 


a:  =  — 


b  'S,-K' 


In  this  equation  s  and  b  represent  the  volumes  of  salt  solution  and  blood  mixed. 
*S'  represents  the  specific  gravity  of  the  serum  and  salt  solution  obtained  on 
allowing  the  blood-corpuscles  to  settle,  So  the  specific  gravity  of  the  serum,  and 
K  that  of  the  salt  solution. 

For  horse's  blood  two  other  shorter  methods  may  be  made  use  of  (see  the 
original  article). 

Important  objections  have  been  presented  by  several  investigators,  such 
as  Eykivl^n,  Biernacki,  and  Hedin,i  against  the  above  methods,  whose 
value,  therefore,  is  questionable.  The  same  is  also  true  for  another  method, 
suggested  by  St.  Bugarsky  and  Tangl  and  partly  corrected,  in  regard  to 
the  calculations,  by  Stewaet.2  This  method  is  based  upon  a  difference 
in  the  electrical  conductivities  of  the  blood  and  the  plasma.  According  to 
the  investigations  of  P.  Franckel,^  the  results  obtained  by  determii:ing 
the  conductivities  give  the  same  figures  as  those  by  Bleibtreu's  method, 
at  least  for  human,  horse,  ox,  and  dog  bloods.  Stewart  has  also  worked 
out  a  colorimetric  method  for  the  estimation  of  the  volume  of  the  blood- 
corpuscles  and  the  plasma,  which  seems  to  be  worth  applying. 

For  clinical  purposes  the  relative  volume  of  corpuscles  in  the  blood  may 
be  determined  by  the  use  of  a  small  centrifuge  called  a  hcematocrit,  constructed 
bv  Blix  and  described  and  tested  by  Hedin.     A  measured  quantity  of 


1  Biernacki,  Zeitschr.  f.  physiol.  Chem.,  19;   Eykman,  Pfl  ger's   Arch.,  60;    Hedin, 
ibid.,  and  Skand.  Arch.  f.  Physiol.,  o. 

2  Bugarsky  and  Tangl,  Centralbl.  f.  Physiol.,  11;    Stewart,  Journ.  of  Physiol.,  24. 
2  Franckel,  Zeitschr.  f.  klin.  Med.,  52. 


ANALYTICAL   METHODS.  237 

blood  is  mixed  with  a  known  volume  (best  an  equal  volume)  of  a  fluid 
which  prevents  coagulation.  This  mixture  is  introduced  into  a  tube  and 
then  centrifuged.  According  to  Hedin  it  is  best  to  treat  the  blood,  which 
is  kept  fluid  by  1  p.  m.  oxalate,  with  an  equal  volume  of  a  9  p.  m.  NaCl 
solution.  After  complete  centrifugalization,  the  layer  of  blood-corpuscles  is 
read  off  on  the  graduated  tube  and  the  volume  of  blood-corpuscles  (or  more 
correctly  the  layer  of  blood-corpuscles)  in  100  vols,  of  the  blood  calculated 
therefrom.  By  means  of  comparative  counts,  Hedin  and  Daland  have 
found  that  an  approximately  constant  relation  exists  between  the  volume 
of  the  layer  of  blood-corpuscles  and  the  number  of  red  corpuscles  under 
physiological  conditions,  so  that  the  number  of  corpuscles  may  be  calculated 
from  the  volume.  Daland  ^  has  shown  that  such  a  calculation  gives 
approximate  results  also  in  disease,  when  the  size  of  the  blood-corpuscles 
does  not  essentially  deviate  from  the  normal.  In  certain  diseases,  such  as 
pernicious  ansemia,  this  method  gives  such  inaccurate  results  that  it  cannot 
be  used. 

KoppE^  has  recently  shown  that  in  centrifuging  blood  very  rapidly, 
more  than  5000  times  per  minute,  the  blood-corpuscles  may  be  so 
completely  separated  that  all  intermediate  fluid  is  removed.  Because 
of  the  absence  of  this  intermediate  fluid  the  refraction  is  changed;  the 
outer  layers  of  the  erythrocytes  containing  fat  become  transparent,  and 
the  column  of  blood-corpuscles  becomes  transparent  and  laky.  If  the 
volume  of  the  separated  column  of  blood-corpuscles  is  determined  and 
the  number  of  red  blood-corpuscles  counted,  the  absolute  volume  of  these 
latter  can  be  determined  by  this  method. 

In  determining  the  relationship  between  the  weight  of  blood-corpuscles 
and  the  weight  of  blood-fluid,  we  generally  proceed  in  the  following  manner: 

If  any  substance  is  found  in  the  blood  which  belongs  exclusively  to  the 
plasma  and  does  not  occur  in  the  blood-corpuscles,  then  the  amount  of 
plasma  contained  in  the  blood  may  be  calculated  if  we  determine  the  amount 
of  this  substance  in  100  parts  of  the  plasma  or  serum  respectively  on  one 
side,  and  in  100  parts  of  the  blood  on  the  other.  If  we  represent  the  amount 
of  this  substance  in  the  plasma  by  f  and  that  in  the  blood  by  h,  then  the 

amount  of  x  in  the  plasma  from  100  parts  of  blood  is  x= '—. 

Such  a  substance,  which  occurs  only  in  the  plasma,  is  fibrin  according 
to  Hoppe-Seyler,  sodium  according  to  Bunge  (in  certain  kinds  of  blood), 
and  sugar  according  to  Otto.-^  The  experimenters  just  named  have  tried 
to  determine  the  amount  of  the  plasma  and  blood-corpuscles,  respectively, 
in  different  kinds  of  blood,  starting  from  the  above-mentioned  substances. 

Another  method  suggested  by  Hoppe-Seyler  is  to  determine  the  total 
amount  of  haemoglobin  and  proteins  in  a  portion  of  blood,  and  on  the  other 
hand  the  amount  of  haemoglobin  and  proteins  in  the  blood-corpuscles  (from 
an  equal  portion  of  the  same  blood)  which  have  been  sufficiently  washed 
with  common-salt  solution  by  centrifugal  force.     The  figure  obtained  as  a 

'  Hedin,  Skand.  Arch.  f.  Physiol.,  2,  134  and  361,  and  5;  Pfliiger's  Arch.,  60; 
Daland,  Fortschritte  d.  Med.,  9. 

2  Pfliiger's  Arch.,  107. 

'Hoppe-Seyler,  Handb.  d.  physiol.  u.  path.  chem.  Analyse,  7.  Aufl.;  Bunge,  Zeit- 
fichr.  f.  Biologic,  12;  Otto,  Pfliiger's  Arch.,  35. 


238  THE  BLOOD. 

difference  between  these  two  determinations  corresponds  to  the  amount  of 
proteins  which  was  contained  in  the  serum  of  the  first  portion  of  blood. 
If  we  now  determine  the  proteins  in  a  special  portion  of  serum  of  the  same 
blood,  then  the  amount  of  senmi  in  the  blood  is  easily  determined.  The 
usefulness  of  this  method  has  been  confirmed  by  Bunge  by  the  control 
experiments  with  the  sodium  determinations.  If  the  amount  of  serum  and 
blood-corpuscles  in  the  blood  is  known,  and  we  then  determine  the  amount 
of  the  different  blood-constituents  in  the  blood-serum  on  one  side  and  of 
the  total  blood  on  the  other,  the  distribution  of  these  different  blood- 
constituents  in  the  two  chief  components  of  the  blood,  plasma  and  blood- 
corpuscles,  may  be  ascertained.  In  the  table  opposite  are  given  analyses 
of  the  blood  of  various  animals  by  Abderhalden  ^  according  to  Hoppe-Sey- 
ler's  and  Bunge's  methods.  The  analyses  of  human  blood  by  C.  Schmidt  ^ 
are  older  and  were  made  according  to  another  method,  hence  perhaps  the 
results  for  the  weights  of  the  corpuscles  are  a  little  too  high.  All  the  results 
are  in  parts  per  1000  parts  of  blood. 

The  relation  between  blood-corpuscles  and  plasma  may  vary  considerably 
under  different  circumstances  even  in  the  same  species  of  animal.  In 
animals,  in  most  cases  considerably  more  plasma  is  found,  sometimes  twa 
thirds  of  the  weight  of  the  blood.^  For  human  blood  Arronet  has  found 
478.8  p.  m.  blood-corpuscles  and  521.2  p.  m.  seiaun  (in  defibrinated  blood) 
as  an  average  of  nine  determinations.  Schneider'*  found  349.6  and  650.4 
p.  m.  respectively  in  women. 

The  sugar  occurs,  it  seems,  only  in  the  serum  and  not  with  the  blood- 
corpuscles.  The  same  is  true,  according  to  Abderhalden,  for  the  lime, 
fat,  and  perhaps  also  the  fatty  acids.  The  small  traces  of  bile-acids  found 
in  normal  blood  are,  according  to  Croftan,^  contained  in  the  leucocytes. 
The  division  of  the  alkalies  between  the  blood-corpuscles  and  the  plasma 
is  different,  as  the  blood-corpuscles  from  the  pig,  horse,  and  rabbit  contain 
no  soda,  those  from  human  blood  are  richer  in  potassium,  and  the  corpuscles 
from  0X-,  sheep-,  goat-,  dog-,  and  cat-blood  are  considerably  richer  in 
sodium  than  potassium.  Chlorine  exists  in  all  blood  to  a  greater  extent 
in  the  serum  than  in  the  blood-corpuscles.  Iodine  is  found  only  in  the 
senmi,  while  iron  occurs  chiefly  in  the  form-elements,  especially  in  the 
erythrocytes.  As  the  iiucleoproteids  contain  iron,  some  iron  always  occurs 
in  the  leucocytes,  and  traces  of  iron  are  also  found  in  the  serum.  This 
amount  under  normal  conditions  is  very  small,  while  in  disease  the  relation 
Ijetween  hsemoglobin-iron  and  other  blood-iron  does  not  seem  to  change 

'  Zeitschr.  f.  physiol.  Chem.,  23  and  25. 

^  Cited  and  in  part  recalculated  from  v.  Gorup-Besanez,  Lehrb.  d.  physiol.  Chem., 
4.  Aufl.,  345. 

'See  Sacharjin  in  Hoppe-Seyler's  Physiol.  Chem.,  447;  Otto,  Pfluger's  Arch.,  35; 
Bun<re,  1.  c;   L.  and  M.  Bleibtreu,  Pfliiger's  Arch.,  51. 

■•  Arronet,  Maly's  Jahresber.,  17;   Schneider,  Centrulbl.  f.  Physiol.,  5,  362. 

*Pfliiger'sArch.,90. 


COMPOSITION   OF  THE   BLOOD. 


239 


Water 

Solids 

Harnoglobin 

Proteid 

Sugar 

Cholesterin 

Lecithin 

Fat 

Fatty  acids 

Phosphoric    acid  ( 
as  nuclein  ' 

Soda. 

Potash 

Iron  oxide 

Lime. 

Magnesia 

Chlorine 

Phosphoric  acid.  . 
Inorganic  P2O5.  .  . 


Pig-blood. 


_S    -  T 
ffl 

272.20 

162.S9 

142.20 

8.35 

0.213 
1.504 

0.027 
0.0455 


2.1.57 
0.696 

0.06.56 
0.642 
0.89.56 
0.7194 


518.36 
46.. 54 

38.26 
0.684 
0.231 
0.805 
1.104 
0.448 

0.0123 

2.401 
0.152 

0.0689 

0.0233 

2.048 

0.1114 

0.0296 


0.\-blood. 


5 

192.65 

132.85 

103.10 

20.89 

1.100 
1.220 


0.0178 

0.7266 
0.2351 
0.544 

0.0056 
0.5901 
0.2392 
0.1140 


616.25 

58.249 

48.901 

0.708 

0.835 

1.129 

0.625 

0.0089 

2.9084 
0.1719 

0.0805 
0.0300 
2.4889 
0.1646 
0.0571 


Horse-blood. 


243.87 
153.84 
125.8 
20.05 

0.26 
1.93 

0.02 
0.05 


1.32 
0.59 

0.04 
0.18 
0.98 
0.76 


551.14 
51.15 

42.65 
0.90 
0.31 
1.05 
0.50 
0.36 

0.01 

2.62 
0.15 

0.07 
0.03 
2.20 
0.15 
0.05 


Dog-blood. 


a 

277.71 
165.10 
145.6 
2.36 

0.56 
1.02 


0.05 

1.27' 
O.lll 
0.71 

0.03' 
0.60 
0.67 
0.54 


514.30 
42.89 

34.05 
0.74 
0.37 
0.98 
0.91 
0.70 

0.01 

2.39 
0.14 

0.06 
0.03 
2.31 
0.14 
0.05 


Bull-blood. 


206.81 

127.50 

106.40 

15.38 

0.610 
0.953 


608.03 
57.66 

46.41 
0.679 
0.599 
1.244 
2.3.57 
0.494 

0.0194  0.0089 

0.839 
0  233 
0.562 


0.009 
0.628 
0.236 
0.133 


2.873 
0.174 


0.073 
0.027 
2.453 
0.156 
0.041 


Sheep-blood. 


s 

200.39 

118.82 

102.80 

12.80 

1.147 
1.329 


0.760 
0.236 
0.545 

0.006 
0.575 
0.228 
0.088 


Ed 

300 


624  1ft 
56.63 

.46.5& 

0.708 

0.891 

1.088 

0.859 

0.490S 

0.0235  O.OIO* 


2.917 
0.172 

:0.089 
0.027 
,2.516 
0.163 
0.057 


Goat-blood. 

Cat-blood. 

Rabbit-blood. 

Human  Blood, 
Man. 

Human  Blood, 
Woman. 

o; 

lU 

<u 

V 

(U 

0 

c 

M(M 

0 

,   =M 

.00 

1  S® 

.0 

3  — 

.a> 

.  2° 

-OJ 

,    3n 

-t- 

£<N 

~  "'t 

Ed 

Tl&ci 

£t^ 

TJ&C^ 

Ed 

~  Ceo 

£co 

C  Of 

3  ^'^ 

C  0^5 

3--0 

0  0I> 

3'=a 

2  o-i 

300 

2  ^=^ 

C  0  ?0 

-0 

0  -.i^ 

-0 

0  ccc 

-co 

0  oiO 

>--r 

c  on 

Co 

a 

■Jl 

H 

rjl 

« 

m 

« 

m 

n 

XJl 

Water 

211.35 

135.86 

592.54 
60.25 

270.90 
163.11 

524.17 
41.35 

235.74 
136  37 

518.18 
46.71 

349.69 
163.33 

439.02 
47.96 

272  56 
123.68 

551. 9» 

Solids 

51.77 

Haemoglobin 

11 2.. 50 

— 

143.2 

— 

123.50 

— 

Proteid 

18.76 
0.601 

50.96 
0.822 
0.698 

11.62 
0.5.56 

33.16 
0.860 
0.3.39 

4.55 
0.268 

33  63 
1.036 
0.343 

Organic 
bodies 

Cholesterin 

Lecithin  .     

1.339 

1.127 

1.3.54 

0.971 

1.722 

1.105 

159.59 

43.82 

120.13 

46.70 

Fat 

— 

0.0407 
0.398 

— 

0.446 
0.282 

z 

0.749 
0.507 

Fatty  acids 

Inorg. 

Phosphoric   acid  | 

0.028 

0.0117 

0.063 

0.009 

0.040 

0.015 

3.74 

4.14 

3.55 

5.07 

Soda 

0.7.55 

2.824 

1.174 

2  512 

— 

2.789 

0.24 

1.66 

0.65 

1.92 

Potash.      

0.236 

0.160 

0.112 

0.148 

1  946 

0.162 

1.59 

0.15 

1.41 

0  20 

Iron  oxide    ...... 

0  547 

— 

0.694 

— 

0.615 

. — 

. — 

- — 

— 

— 

Lime 

— 

0.078 

— 

0.062 

— 

0.072 

— 

— 

— 

— 

Magnesia 

0  014 

0.026 

0  035 

0.024 

0  029 

0.028 

— 

— 

— 

— 

Chlorine 

0.514 

2  409 

0.455 

2  360 

0.460 

2.438 

0.90 

1.72 

0.36 

0-14 

Phosphoric  acid.  . 

0.243 

0.1.54 

0.697 

0.133 

0.835 

0.151 

— 

— 

— 

— 

Inorganic  PoOs.  .  ■ 

0.097 

0.045 

0.515 

0.040 

0.645 

0.040 

— 

— 

very  much.  There  are  also  found  in  the  blood  manganese  and  traces  of 
lithium,  copper,  lead,  silver,  and  in  menstrual  blood  arsenic  has  also  been 
noted.  The  blood  as  a  whole  contams  in  ordinary'  cases  770-820  p.  m. 
water,  with  180-230  p.  m.  solids;  of  these  173-220  p.  m.  are  organic  and 
6-10  p.  m.  inorganic.  The  organic  consists,  deducting  6-12  p.  m.  of  extrac- 
tive bodies,  of  proteins  and  haemoglobin.  The  amount  of  this  last-men- 
tioned body  in  human  blood  is  about  130-150  p.  m.  In  the  dog,  cat;  pig. 
and  horse  the  quantity  of  haemoglobin  is  about  the  same,  but  is  lower  in 
the  Ijlood  from  the  ox,  bull,  sheep,  goat^  and  rabbit  (Abderhalden). 


240  THE  BLOOD. 

The  amount  of  sugar  in  the  blood  is  on  an  average  1-1.5  p.  m.  It 
seems  to  be  independent  of  the  composition  of  the  food,  but  feeding 
-with  large  amounts  of  sugar  or  dextrin  causes  a  considerable  increase  in 
the  sugar  of  the  blood,  as  observed  by  Bleile.  When  the  quantity  of 
sugar  amounts  to  more  than  3  p.  m.,  then,  according  to  Cl.  Bernard,^ 
sugar  occurs  in  the  urine,  and  a  glycosuria  appears.  An  increase  in  the 
quantity  of  sugar  takes  place,  as  first  observed  by  Bernard  and  lately 
substantiated  by  Fr.  Schenck,  after  removal  of  blood.  According  to 
Henriques^  this  increase  of  the  reducing  power,  at  least  in  dogs,  is  not 
due  to  sugar,  but  chiefly  to  jecorin,  which  substance  is  the  cause  of  more  of 
the  reduction  in  normal  blood  than  the  sugar.  It  is  difficult  to  judge  of  the 
value  of  many  statements  as  to  the  amount  of  sugar  and  the  reducing 
power  of  the  blood,  because  the  experimenters  generally  have  not  con- 
sidered the  presence  of  a  certain  quantity  of  jecorin  or  conjugated  glu- 
curonic acids,  or  they  were  unable  to  detect  them. 

The  quantity  of  urea,  which,  according  to  Schondorff,  is  equally  divided 
between  the  blood-corpuscles  and  the  plasma,  is  greater  on  taking  food  than 
in  starvation  (Gr^hant  and  Quinquaud,  Schondorff)  and  varies  between 
0.2  and  1.5  p.  m.  In  dogs  Schondorff  found  in  starvation  a  minimum 
of  0.348  p.  m.  and  a  maximum  of  1.529  p.  m.  at  the  point  of  highest 
urea  formation.  Gottlieb  obtained  much  lower  results  by  another  direct 
method,  namely,  in  starvation  0.1-0.2,  and  after  meat  feeding  0.28-0.56 
p.  m.  In  man  v.  Jaksch  ^  found  0.5-0.6  p.  m.  urea  in  normal  blood.  The 
quantity  of  urea  is  somewhat  increased  in  fever,  and  in  general  in  augmented 
protein  metabolism  and  the  increased  urea  formation  depending  thereon. 
A  more  important  mcrease  in  the  quantity  of  urea  in  the  blood  occurs  in  a 
retarded  elimination  of  urea,  as  in  cholera,  also  in  cholera  infantum  and 
in  infections  of  the  kidneys  and  the  urinary  passages.  After  ligaturing 
the  ureters  or  after  extirpation  of  the  kidneys  of  animals,  an  accumulation 
of  urea  takes  place  in  the  blood. 

V.  Schroder  first  showed  that  the  blood  of  the  shark  was  very  rich  in 
urea,  and  the  quantity  indeed  amounted  to  26  p.  m.  Baglioni*  has  re- 
cently shown  that  this  large  quantity  of  urea  is  of  the  greatest  importance, 
as  the  presence  of  urea  in  these  animals  is  a  necessary  life-condition  for 
the  heart  and  very  probably  for  all  organs  and  tissues. 

'  Bleile,  Arch.  f.  (Anat.  u.)  Physiol.,  1879;  Bernard,  Lemons  sur  le  diab^te,  Paris, 
1877. 

'  Schenck,  Pfliiger's  Arch.,  57;  Henriques,  Zeitschr.  f.  physiol.  Chem.,  23,  See  also 
Kolisch  and  Stejskal,  Wien.  klin.  Wochenschr.,  1898. 

'  Gr^hant  et  Quinquaud,  Joum.  de  I'anatomie  et  de  la  physiol.,  20,  and  Compt. 
rend.,  98;  Schondorff,  Pfliiger's  Arch.,  54  and  63j  Gottlieb,  Arch.  f.  exp.  Path.  u. 
Pharm.,  42;  v,  Jaksch,  Leyden-Festschr.,  I,  1901. 

*  V.  Schroder,  Zeitschr.  f.  physiol.  Chem.,  14;  Baglioni,  Centralbl.  f.  Physiol.,  19. 


COMPOSITION  OF  THE  BLOOD.  241 

The  blood  also  contains  traces  of  ammonia.  According  to  Horodynski, 
Salaskin,  and  Zaleski.^  who  worked  with  the  improved  Nencki  and 
Zaleski  method,  the  quantity  in  arterial  dog-blood  was  0.41  milligram  in 
100  grams  of  blood.  The  blood  of  the  portal  vein  contains  considerably 
more  than  the  blood  of  the  arteries,  being  3-4.5  times  richer;  this  is 
disputed  by  Biedl  and  Wixterberg,^  however.  The  blood  from  healthy 
persons  contains  on  an  average  0.90  milligram  per  100  c.c,  according  to 
WiNTERBERG.3  The  quantity  of  uric  acid  may  be  0.1  p.  m.  in  bird's  blood 
(v.  Schroder'*).  Uric  acid  has  not  been  detected  with  positiveness  in 
human  blood  under  normal  conditions,  while  it  has  been  found  in  the 
blood  in  gout,  croupous  pneumonia,  and  certain  other  diseased  conditions. 
Lactic  acid  was  first  found  in  human  blood  by  Salomon  and  then  by 
Gaglio,  Berlinerblau,  and  Irisawa.  The  quantity  of  lactic  acid  may 
vary  considerably.  Berlinerblau  found  0.71  p.  m.  as  maximum. 
Saito  and  K^tsuyama  ^  found  on  an  average  0.269  p.  m.  in  hen's  blood, 
and  after  carbon-monoxide  poisoning  the  quantity  increased  to  1.227  p.  m. 

The  Composition  of  the  Blood  in  Different  Vascular  Regions  and  under 

Different  Conditions. 

Arterial  and  Venous  Blood.  The  most  striking  difference  between 
these  two  kinds  of  blood  is  the  variation  in  color  caused  by  their  containing 
different  amounts  of  gas  and  different  amounts  of  oxyhsemoglobin  and 
haemoglobin.  The  arterial  blood  is  light  red;  the  venous  blood  is  dark  red, 
dichroitic,  greenish  by  transmitted  light  through  thin  laj-ers.  The  arterial 
coagulates  more  quickly  than  the  venous  blood.  The  latter,  on  account  of 
the  transudation  which  takes  place  in  the  capillaries,  was  formerly  said  to 
be  somewhat  poorer  in  water  but  richer  in  blood-corpuscles  and  haemo- 
globin than  the  arterial  blood ;  but  this  is  denied  by  modern  investigators. 
According  to  Kruger  ®  and  his  pupils  the  quantity  of  dr\^  residue  and 
ha?moglobin  in  blood  from  the  carotid  artery  and  from  the  jugular  vein  (in 
cats)  is  the  same.  Rohmann  and  Muhsam  ^  could  not  detect  any  differ- 
ence in  the  quantity  of  fat  in  arterial  and  venous  blood. 

Blood  jrom  the  Portal  Vein  and  the  Hepatic  Vein.  In  consequence  of 
the  small  quantities  of  bile  and  lymph  found  relatively  to  the  large  quantity 

^  Zeitschr.  f.  physiol.  Chem.,  35,  which  also  gives  the  older  literature. 

»Pfliiger's  Arch.,  88. 

'  Wien.  klin.  Wochenschr.,  1897,  and  Zeitschr.  f.  klin.  Med.,  35. 

*  Ludwig's  Festschrift,  1887. 

*  Irisawa,  Zeitschr.  f.  physiol.  Chem.,  17,  which  also  gives  the  older  literature;  Saito 
and  Katsuyama,  ihid.,  32. 

°  Zeitschr.  f .  Biologie,  26.     This  also  gives  the  literature  on  the  composition  of  the 
blood  in  different  vascular  regions. 
'  Pfliiger's  Archiv,  46. 


242  THE  BLOOD, 

of  blood  circulating  through  the  liver  in  a  given  time^  we  can  hardly  expect 
to  detect  by  chemical  analysis  a  positive  difference  in  the  composition 
between  the  blood  of  the  portal  and  hepatic  veins.  The  statements  in 
regard  to  such  a  difference  are  in  fact  contradictory.  For  example,  Dros- 
DOFF  has  found  more  haemoglobin  in  the  hepatic  than  in  the  portal  vein^ 
while  Otto  found  less.  Kruger  finds  that  the  quantities  of  haemoglobin^ 
as  well  as  of  the  solids,  in  the  blood  from  the  vessels  passing  to  and  from 
the  liver  are  different,  but  a  constant  relationship  cannot  be  determined. 
The  disputed  question  as  to  the  varying  quantities  of  sugar  in  the  portal 
and  hepatic  veins  will  be  discussed  in  a  following  chapter  (see  Chapter 
VIII,  on  the  formation  of  sugar  in  the  liver).  After  a  meal  rich  in  carbo- 
hydrates, the  blood  of  the  portal  vein  not  only  becomes  richer  in  dextrose^ 
but  may  contain  also  dextrin  and  other  carbohydrates  (v.  Mering,  Otto  *), 
The  amount  of  urea  in  the  blood  from  the  hepatic  vein  is  greater  than 
in  other  blood  (Greihant  and  Quinquaud^).  In  regard  to  the  quantity 
of  ammonia,  see  page  241. 

Blood  of  the  Splenic  Vein  is  decidedly  richer  in  leucocytes  than  the 
blood  from  the  splenic  artery.  The  red  blood-corpuscles  of  the  blood  from 
the  splenic  vein  are  smaller  than  the  ordinary,  less  flattened,  and  show  a 
greater  resistance  to  water.  The  blood  from  the  splenic  vein  is  also  claimed 
to  be  richer  in  water,  fibrin,  and  protein  than  the  ordinary  venous  blood. 
According  to  v.  Middendorff,  it  is  richer  in  haemoglobin  than  arterial 
blood.  KriIger  ^  and  his  pupils  have  found  that  the  blood  from  the  vena 
lienalis  is  generally  richer  in  haemoglobin  and  solids  than  arterial  blood; 
still  the  contrary  is  often  found.  The  blood  from  the  splenic  vein  coagu- 
lates slowly. 

The  Blood  from  the  Veins  of  the  Glands.  The  blood  circulates  with 
greater  rapidity  through  a  gland  during  acti\aty  (secretion)  than  when  at 
rest,  and  the  outflowing  venous  blood  has  therefore  during  activity  a  lighter 
red  color  and  a  greater  amount  of  oxygen.  Because  of  the  secretion  the 
venous  blood  also  becomes  somewhat  poorer  in  water  and  richer  in  solids. 

The  blood  from  the  Muscidar  Veins  shows  an  opposite  behavior,  for 
during  activity  it  is  darker  and  more  venous  in  its  properties  because  of  the 
increased  absorption  of  oxygen  by  the  muscles  and  still  greater  production 
of  carbon  dioxide  than  when  at  rest. 

Menstrual  Blood,  according  to  an  old  statement,  has  not  the  power  of 
coagulating.  This  statement  is  nevertheless  false,  and  the  apparent  un- 
coagulability  depends  in   part  on  the  retention    of  the  blood-clot  by  the 

'  Drosdoff,  Zeitschr.  f.  physiol.  Chem.,  1;   Otto,  Maly's  Jahresber  ,17;    v.  Mering, 
Arch,  f.  (Anat.  u.)  Physiol ,  1877,  214. 
M    c. 
'v.  Middendorff,  Centralbl.  1.  Physiol.,  2,  753;    Kriiger,  1.  c. 


COMPOSITION  OF  THE  BLOOD.  243 

womb  and  the  vagina,  so  that  only  fluid  cruor  is  at  times  eliminated,  and 
in  part  on  a  contamination  with  vaginal  mucus,  which  disturbs  the  coagu- 
lation. Menstrual  blood,  according  to  Gautier  and  Bourcet,  contains 
arsenic  and  is  also  richer  in  iodine  than  other  blood  (see  blood-senmi, 
page  187). 

The  Blood  of  the  Two  Sexes.  Woman's  blood  coagulates  somewhat  more 
quickly,  has  a  lower  specific  gravity,  a  greater  amount  of  water,  and  a 
smaller  quantity  of  solids  than  the  blood  of  man.  The  amount  of  blood- 
corpuscles  and  ha3moglobin  is  somewhat  smaller  in  woman's  blood.  The 
amount  of  haemoglobm  is  146  p.  m.  for  man's  blood  and  133  p.  m.  for 
woman's. 

During  pregnancy  Nasse  has  observed  a  decrease  in  the  specific  gravity, 
with  an  increase  in  the  amount  of  water,  until  the  end  of  the  eighth  month. 
From  then  the  specific  gravity  increases,  and  at  deliverj^  it  is  normal  again. 
The  amount  of  fibrin  is  somewhat  increased  (Becquerel  and  Rodier, 
Nasse).  The  number  of  blood-corpuscles  seems  to  decrease.  In  regard  to 
the  amount  of  haemoglobin  the  statements  are  somewhat  contradictory. 
CoHNSTEiN  found  the  number  of  red  corpuscles  diminished  in  the  blood 
of  pregnant  sheep  as  compared  with  non-pregnant,  but  the  red  corpuscles 
were  larger  and  the  quantity  of  haemoglobin  in  the  blood  was  greater  in  the 
first  case.  Mollenberg  ^  found  in  most  cases  an  increase  in  the  amount 
of  haemoglobin  m  pregnancy  in  the  last  months. 

The  Blood  at  Different  Periods  of  Life.  Foetal  and  infant  blood  is  richer 
in  erythrocytes  and  haemoglobin  than  the  blood  of  the  mother.  The 
highest  percentage  of  haemoglobin  in  the  blood  has  been  observed  l^y 
several  investigators,  such  as  Cohnstein  and  Zuntz,  Otto,  Winternitz, 
Abderhalden,  Schwinge,  and  others,  immediately  or  very  soon  after 
birth  or  at  least  ^vithin  the  first  few  days.  In  man,  two  or  three  days  after 
birth  the  haemoglobin  reaches  a  maximum  (200-210  p.  m.)  which  is  greater 
than  at  any  other  period  of  life.  This  is  the  cause  of  the  great  abundance 
of  solids  in  the  blood  of  new-bom  infants,  as  observed  by  several  inves- 
tigators. The  quantity  of  haemoglobin  and  blood-corpuscles  sinks  gradu- 
ally from  this  first  maximum  to  a  minimum  of  about  110  p.  m.  haemoglobin, 
which  minimum -appears  in  human  beings  between  the  fourth  and  eighth 
years.  The  quantity  of  haemoglobin  then  increases  again  until  about  the 
twentieth  year,  when  a  second  maximum  of  137-150  p.  m.  is  reached.  The 
haemoglobin  remains  at  this  point  only  to  about  the  forty-fifth  year,  and 
then  gradually  and  slowly  decreases  (Leichtenstern,  Otto  2).     According 

'  Nasse,  Maly's  Jahresber.,  7;  Becquerel  and  Rodier,  Traite  de  chim.  pathol., 
Paris.  1854;  Cohnstein,  Pfli.ger's  Arch.,  34,'  233;  Mollenberg,  Maly's  Jahresber.,  31, 
1S5.     See  also  Payer,  Arch.  f.  Gynak.,.  71. 

■  Cohnstein  and  Zuntz,  Pfliiger's  Arch.,  34;  Winternitz.  Zeitschn-f.  physiol.  Chem., 
22;   Leichtenstern,  Untersuch.  iiber  den  Hamoglobingehalt  des  Blutes,  etc.,  Leipzig, 


244  THE  BLOOD. 

to  older  statements,  the  blood  at  old  age  is  poorer  in  blood-corpuscles  and 
protein  bodies,  but  richer  in  water  and  salts. 

The  Influence  of  Food  on  the  Blood.  In  complete  starvation,  no  decrease 
in  the  amount  of  solid  blood-constituents  is  found  to  take  place  (Panum 
and  others).  The  amount  of  hiemoglobin  is  increased  a  little,  at  least  in 
the  early  period  (Subbotix,  Otto,  Hermann  and  Groll,  Luciani  and 
BuFALiNi),  and  also  the  number  of  red  blood-corpuscles  increases  (Worm 
MuLLER,  BuNTZEN^),  which  probably  depends  partly  on  the  fact  that  the 
blood-corpuscles  are  not  so  quickly  transformed  as  the  serum  and  partly 
on  a  greater  concentration  due  to  loss  of  water.  In  rabbits  and  to  a  less 
extent  in  dogs,  Popel  found  that  complete  abstinence  had  a  tendency 
to  increase  the  specific  gravity  of  the  blood.  The  amount  of  fat  in  the 
blood  may  be  somewhat  increased  in  starvation  because  the  fat  is  taken 
up  from  the  fat  deposits  and  carried  to  the  various  organs  by  the  blood 
(N.  SCHULZ,  Daddi^). 

After  a  rich  meal  the  relative  number  of  blood-corpuscles,  after  secretion 
of  digestive  juices  or  absorption  of  nutritive  liquids,  may  be  increased  or 
diminished  (Buntzen,  Leichtenstern).  The  number  of  white  blood- 
corpuscles  may  be  considerablv  increased  after  a  diet  rich  in  proteins. 
After  a  diet  rich  in  fat  the  plasma  becomes,  even  after  a  short  time,  more 
or  less  milky -white,  like  an  emulsion.  The  composition  of  the  food  acts 
essentially  on  the  amount  of  haemoglobin  in  the  blood.  The  blood  of 
herbivora  is  generally  poorer  in  haemoglobin  than  that  of  camivora, 
and  SuBBOTiN  has  observed  in  dogs  after  a  partial  feeding  with  food  rich 
in  carbohydrates  that  the  amount  of  hsemoglobin  sank  from  the  physio- 
logical average  of  137.5  p.  m.  to  103.2-93.7  p.  m.  Tsuboi^  has  also  shown 
in  experiments  on  rabbits  and  dogs  that  with  an  insufficient  diet  of  bread 
and  potatoes,  where  the  body  gave  up  protein  and  contained  relatively 
much  carl)ohydrate,  the  amount  of  htemoglobin  decreased  and  the  blood 
became  richer  in  water.  According  to  Leichtenstern,  a  gradual  increase 
in  the  amount  of  hsemoglobin  is  found  to  take  place  in  the  blood  of 
himian  beings  on  enriching  the  food,  and  according  to  the  same  inves- 
tigator the  blood  of  lean  persons  is  generally  somewhat  richer  in  h<Tmo- 

1878;  Otto,  Maly's  Jahresber.,  15  and  17;  Abderhalden,  Zeitschr.  f.  physiol.  Chem., 
34;  Schwinge,  Pfliiger's  Arch.,  73  (literature).  See  also  Fehrsen,  Journ.  of  Physiol., 
30.' 

^  Panum,  Virchow's  Arch.,  29;  Subbotin,  Zeitschr.  f.  Biologie,  7;  Otto,  1.  c;  Worm 
Muller.  Transfusion  und  Plethora,  Christiania,  1875;  Buntzen.  see  Maly's  Jahresber., 
9;  Hermann  and  Groll,  Pfliiger's  Arch.,  43;  Luciani  and  Bufalini,  Maly's  Jahresber., 
12. 

'  Popel,  Arch,  des  scienc.  biol.  de  St.  Petersbourg,  4,  354;  Schulz,  Pfliiger's  Arch., 
65;  Daddi,  Maly's  Jahresber.,.  30. 

^Subbotin,  1.  c;  Tsuboi,  Zeitschr.  i.  Biologie,  44. 


INCREASE    IX    THE    RED    CORPUSCLES.  245 

globin  than  blood  from  fat  ones  of  the  same  age.  The  addition  of  iron 
salts  to  the  food  greatly  influences  the  number  of  blood-corpuscles  and 
especially  the  amoimt  of  haemoglobin  they  contain.  The  action  of  the  iron 
salts  is  obscure.i  There  does  not  seem  to  be  any  doubt  that  not  only  is 
the  iron  contained  in  the  food  m  the  form  of  organic  compoimds  active, 
but  also  iron  salts  and  therapeutic  iron.  Accordmg  to  Buxge  and  his 
pupils  the  iron  preparations  only  act  indirectly.  They  may  combine  with 
the  sulphuretted  hydrogen  of  the  intestinal  canal  and  thereby  prevent  the 
iron  associated  in  the  food  as  assimilable  protem  compounds  from  being 
eliminated  as  iron  sulphide  (Bunge),  or  they  may  perhaps  act  as  excitants 
upon  the  blood-forming  organs  (Abderhaldex). 

An  increase  in  the  number  of  red  corpuscles,  a  true  "plethora  poly- 
cythcemia,"  takes  place  after  transfusion  of  blood  of  the  same  species  of 
animal.  According  to  the  observations  of  Paxum  and  Worm  Muller.^ 
the  blood-liquid  is  quickly  eliminated  and  transformed  in  this  case — the 
water  being  eliminated  principally  by  the  kidneys  and  the  protein  burned 
into  urea,  etc. — while  the  blood-corpuscles  are  preserved  longer  and  cause 
a  " polycijthcvmia."  A  relative  mcrease  in  the  number  of  red  corpuscles 
is  found  after  abimdant  transudation  from  the  blood,  as  in  cholera  and 
heart-failure  •wdth  considerable  congestion.  An  mcrease  in  the  number 
of  red  blood-corpuscles  has  also  been  observed  under  the  influence  of 
diminished  pressure  or  m  high  altitudes.  Viault  first  called  attention  to 
the  fact  that  the  number  of  red  corpuscles  was  ver\-  great  in  the  blood  of 
man  and  animals  living  in  high  regions.  According  to  him  the  llama  has 
about  16  million  blood-corpuscles  per  cubic  millimeter.  By  observations 
on  himself  and  others,  as  well  as  on  animals,  Viault  found  the  first  effect 
of  sojourning  in  high  localities  was  a  very  considerable  increase  in  the 
number  of  red  corpuscles,  in  his  ovm.  case  5-^  millions.  A  similar  increase 
of  the  red  blood-corpuscles,  as  also  an  increase  m  the  quantity  of  hemo- 
globin under  the  influence  of  diminished  pressure,  has  been  observed  by 
many  other  investigators  in  human  beings  as  well  as  in  animals.  Investi- 
gators are  not  united  as  to  how  this  increase  is  brought  about.  The  mcrease 
in  the  blood-corpuscles  is  not  absolute  but  is  only  relative,  and  it  is  con- 
sidered by  several  observers  that  there  is  neither  a  new  formation  nor  a 
dimmished  destruction  of  the  blood-corpuscles.  A  relative  increase  may 
be  brought  about  in  different  ways.  For  example,  another  di\'ision  of  the 
blood-corpuscles  in  the  vascular  system  has  been  supposed,  whereby  the 
blood-corpuscles  accumulate  in  the  capillaries,  from  which  region  the  blood 

'  See  Bunge,  Zeitsclir.  f.  physiol.  Chem.,  9;  Hausermann,  ibid.,  23,  where  the  works 
of  Woltering,  Gaule,  Hall,  Hoehhaus,  and  Quincke  are  cited  (the  same  work  con- 
tains a  table  of  the  quantity  of  iron  in  various  foods);  Kunkel,  Pfhiger's  Arch.,  61; 
Macallum,  Journal  of  Physiol.,  16;   Abderhalden,  Zeitsclir.  f.  Biologic,  39. 

'  Panum,  Virchow's  Arch.,  29;    Worm  Midler,  1.  c. 


246  THE  BLOOD. 

has  been  examined  most  often  (Zuntz).  It  is  also  claimed  that  a  con- 
centration of  the  blood  takes  place  by  increased  evaporation  (Grawitz), 
and  finally  an  increase  in  the  blood-corpuscles  has  also  been  explained  by 
assuming  a  contraction  of  the  vascular  system  with  the  pressing  out  of 
plasma  (Bunge,  Abderhalden  i).  In  connection  with  these  experiments,  it 
must  be  remarked  that  several  trustworthy  observations  show  that  under 
the  influence  of  diminished  blood-pressure  an  actual  increase  in  the  red 
blood-corpuscles  takes  place,  and  Zuntz  2  and  his  co-workers  have  also 
shown  that  the  activity  in  the  red  bone-raarrow  is  increased. 

A  decrease  in  the  number  of  red  corpuscles  occurs  in  anaemia  from  differ- 
ent causes.  Every  excessive  hemorrhage  causes  an  acute  anaemia,  or,  more 
correctly,  oligaemia.  Even  during  the  hemorrhage,  the  remaining  blood 
becomes  by  diminished  secretion  and  excretion,  as  also  by  an  abundant 
absorption  of  parenchymous  fluid,  richer  in  water,  somewhat  poorer  in  pro- 
teins, and  strikingly  poorer  in  red  blood-corpuscles.  The  oligaemia  passes 
soon  into  a  hydrsemia.  The  amount  of  protein  then  gradually  increases 
again ;  but  the  re-formation  of  the  red  blood-corpuscles  is  slower,  and  after 
the  hydrsemia  follows  also  an  oligocythaemia.  After  a  little  time  the 
number  of  blood-corpuscles  rises  to  normal;  but  the  re-formation  of  haemo- 
globin does  not  keep  pace  with  the  re-formation  of  the  corpuscles,  and  a 
chlorotic  condition  may  appear.  A  considerable  decrease  in  the  number 
of  red  corpuscles  occurs  also  in  chronic  anaemia  and  chlorosis;  still  in  such 
cases  an  essential  decrease  in  the  amount  of  haemoglobin  occurs  without  an 
essential  decrease  in  the  number  of  blood-corpuscles.  The  decrease  in  the 
amount  of  haemoglobin  is  more  characteristic  of  chlorosis  than  a  decrease 
in  the  number  of  red  corpuscles.  The  statements  on  the  changes  in  the 
blood  in  anaemia  and  chlorosis  differ  very  considerably,  and  in  this  con- 
nection attention  must  be  called  to  the  findings  of  Lorrain  Smith  (based  on 
his  estimation  of  the  oxygen  capacity  and  of  the  blood-volume)  that  in 
chlorosis  an  absolute  diminution  of  the  amount  of  haemoglobin  does  not 
occur,  but,  that  on  the  contrary,  the  total  quantity  of  haemoglobin  may  be 
normal,  with  only  a  relative  diminution  occurring,  due  to  a  pronounced 
increase  of  the  blood-plasma  and  of  the  total  quantity  of  blood.^ 

A  very  considerable  decrease  in  the  number  of  red  corpuscles  (300  000- 
400  000  in  1  c.mm.)  and  diminution  in  the  amount  of  haemoglobin  (J-  ^  ) 
occurs  in  pernicious  anaemia  (Hayem,  Laache,  and  others).  On  the 
contrary,  the  individual  red  corpuscles  are  larger  and  richer  in  haemoglobin 

*  The  literature  on  this  subject  may  be  found  in  Abderhalden,  Zeitschr.  f.  Biologie, 
43;  van  Voornveld,  Pfliiger's  Arch.,  92. 

^  Hohenklima  und  Bergwanderungen,  by  N.  Zuntz,  A.  Loewy,  Franz  M  ller  and  W. 
Caspari,  Berlin,  190G. 

^  Trans.  Path.  Soc.  London,  51,  1900.  Complete  analyses  of  chlorotic  blood  may 
be  found  in  Erben,  Zeitschr.  f.  klin.  Med.,  47. 


NUMBER   OF   LEUCOCYTES.  247 

than  they  ordmarily  are,  and  the  number  stands  in  an  inverse  relationship 
to  the  amount  of  hsemoglobin  (Hayem).  Besides  this  the  red  corpuscles 
often,  but  not  always,  show  in  pernicious  anaemia  remarkable  and  ex- 
traordinary irregularities  of  form  and  size,  which  Quincke  ^  has  termed 
poikilocytosis. 

The  number  of  leucocytes  may,  as  stated  above,  be  increased  under 
physiological  conditions  as  well  as  after  a  meal  rich  in  protein  (physiological 
leucocytosis).  Under  pathological  conditions  a  high  leucocytosis  may  occur, 
and  this  is  especially  found  in  leucaemia,  which  is  characterized  by  a  very 
great  abundance  of  leucocytes  in  the  blood.  The  number  of  leucocytes 
is  markedly  increased  in  this  disease,  and  indeed  not  only  absolutely,  but 
also  in  relation  to  the  number  of  red  blood-corpuscles,  which  are  increased 
to  a  considerable  extent  in  leucaemia.  Leucsemic  blood  has  a  lower  specific 
gravity  than  the  ordinary  blood  (1035-1040),  and  a  paler  color,  as  if  it 
were  mixed  with  pus.  The  reaction  is  alkaline,  but  after  death  it  is  fre- 
quently acid,  probably  due  to  a  decomposition  of  lecithin,  which  is  often 
considerably  increased  in  leucaemia.  Volatile  fatty  acids,  lactic  acid,  glycero- 
phosphoric  acid,  large  amounts  of  xanthine  bodies,  and  so-called  Charcot's 
crystals  (see  semen.  Chap.  XIII)  have  also  been  found  in  leucsemic  blood. 
The  peptone  (proteose)  which  is  found  in  the  leucsemic  blood  after  death, 
and  which  does  not  exist  in  the  fresh  blood,  is,  according  to  Erben,  a 
digestive  product  which  is  produced  by  a  tryptic  enzyme  which  originates 
from  the  leucocytes  as  well  as  by  traces  of  a  peptic  enzyme.  These 
enzymes,  according  to  Erben,  do  not  occur  in  normal  blood,  or  are  so 
firmly  combined  therein  that  on  the  death  of  the  cells  they  are  not  set 
free,  or  at  least  their  action  does  not  become  evident.^ 

A  great  number  of  investigations  have  been  made  on  the  chemical  com- 
position of  blood  in  disease.  But  as  we  have  only  a  few  analyses  of  the 
blood  of  healthy  individuals,  and  as  the  possible  variations  under  physio- 
logical conditions  are  little  known,  it  is  difficult  to  draw  any  positive  conclu- 
sions from  the  analyses  of  pathological  blood.  Unfortunately,  on  account 
of  the  large  number  of  contradictory  statements  of  the  composition  of  the 
blood  of  diseased  human  beings,  it  is  impossible  to  give  a  brief  summary  of 
the  results,  still  the  changes  in  the  blood  in  disease  must  be  of  the  greatest 
importance. 

The  quantity  of  blood  is  indeed  somewhat  variable  in  different  species 
of  animals  and  in  different  conditions  of  the  body;  in  general  we  consider 
the  entire  quantity  of  blood  in  adults  as  about  ^  j\  of  the  weight  of  the 

'  Laache,  Die  Anamie  (Christiania,  1883),  which  also  contains  the  literature; 
Quincke,  Deutsch.  Arch.  f.  klin.  Med.,  20- and  25.  A  complete  chemical  analysis  of 
the  blood  has  been  made  by  Erben,  Zeitschr.  f.  khn.  Med.,  40. 

^  Erben,  Zeitschr.  f.  Heilkunde,  24,  and  Hofmeister's  Beitrage,  S.  See  also  Schumm, 
ibid.,  4  and  5. 


248  THE  BLOOD. 

body,  and  in  new-bom  infants  about  -^.  Haldaxe  and  Lorraix  Smith ,^ 
who  have  determined  the  quantity  of  blood  by  a  new  method,  find  in 
fourteen  persons  that  it  varies  between  ^^  and  gV  of  the  weight  of  the  body. 
Fat  individuals  are  relatively  poorer  in  blood  than  lean  ones.  During 
inanition  the  quantity  of  blood  decreases  less  quickly  than  the  weight  of 
the  body  (Panum^),  and  it  may  therefore  be  also  proportionally  greater 
in  star\dng  individuals  than  in  well-fed  ones. 

By  careful  bleeding  the  quantity  of  blood  may  be  considerably  dimin- 
ished without  any  dangerous  symptoms.  A  loss  of  blood  amounting  to  one 
fourth  of  the  normal  quantity  has  as  a  sequence  no  durable  sinking  of  the 
blood-pressure  in  the  arteries,  because  the  smaller  arteries  accommodate 
themselves  to  the  small  quantities  of  blood  by  contracting  (Worm  ^lijh- 
LER  3).  A  loss  of  blood  amounting  to  one  third  of  the  quantity  reduces  the 
blood-pressure  considerably,  and  a  loss  of  one  half  of  the  blood  in  adults  is 
dangerous  to  life.  The  more  rapid  the  bleeding  the  more  dangerous  it  is. 
New-bom  infants  are  verj-  sensitive  to  loss  of  blood,  and  likewise  fat,  old, 
and  weak  persons  cannot  stand  much  loss  of  blood.  Women  can  stand 
loss  of  blood  better  than  men. 

The  quantity  of  blood  may  be  considerably  increased  by  the  injection  of 
blood  from  the  same  species  of  animal  (Paxum,  Laxdois,  Worm  ]Muller, 
Poxfick).  According  to  Worm  ^Miiller  the  normal  quantity  of  blood  may 
indeed  be  increased  as  much  as  83  per  cent  without  producing  any  abnor- 
mal conditions  or  lasting  high  blood-pressure.  An  increase  of  150  per  cent 
in  the  quantity  of  blood  may,  with  a  considerable  variation  in  the  blood- 
pressure,  be  directly  dangerous  to  life  (Worm  ^Ml'ller).  If  the  quantity 
of  blood  of  an  animal  is  increased  by  transfusion  with  blood  of  the  same 
kind  of  animal,  an  abundant  formation  of  lymph  takes  place.  The  water 
in  excess  is  eliminated  by  the  urine;  and  as  the  protein  of  the  blood-serum 
is  quickly  decomposed,  while  the  red  blood-corpuscles  are  destroyed  much 
more  slowly  (Tschirjew,  Forster,  Paxum,  Worm  ]\1uller4),  a  polycy- 
thsemia  is  gradually  produced. 

The  quantity  of  blood  in  the  different  organs  depends  essentially  on 
their  activity.  During  work  the  exchange  of  material  in  an  organ  is 
more  pronounced  than  during  rest,  and  the  increased  metabolism  is  con- 
nected ■viith  a  more  abundant  flow  of  blood.     Although  the  total  quantity 

'  Joum.  of  Physiol.,  25. 

^  Virchow's  Arch.,  29. 

^  Transfusion  und  Plethora,  Christiania,  1875. 

*  Panum,  Nord.  med.  Ark.,  7;  Virchow's  Arch.,  63;  Landois,  Centralbl.  f.  d.  med. 
Wissensch.,  1875,  and  Die  Transfusion  des  Blutes,  Leipzig,  1875;  Worm  Miiller 
Transfusion  und  Plethora;  Ponfick,  Virchow's  Arch.,  G2;  Tschirjew,  Arbeiten  aus 
der  physiol.  Anstalt  zu  Leipzig,  1874,  292;  Forster,  Zeitschr.  f.  Biologic,  11;  Panum, 
Virchow's  Arch.,  29. 


COMPOSITION  OF  THE  BLOOD.  249 

of  blood  in  the  body  remains  constant,  the  distribution  of  the  blood  in  the 
various  organs  may  be  different  at  different  times.  As  a  rule  the  quantity 
of  blood  in  an  organ  is  an  approximate  measure  of  the  more  or  less 
active  metabolism  going  on  in  the  same,  and  from  this  point  of  view  the 
distribution  of  the  blood  m  the  different  organs  and  groups  of  organs  is  of 
interest.  According  to  Ranke,i  to  whom  we  are  especially  indebted  for 
our  knowledge  of  the  relationship  of  the  acti\dty  of  the  organs  to  the 
quantity  of  blood  contained  therem,  of  the  total  quantity  of  blood  (in  the 
rabbit)  about  one  fourth  comes  to  the  muscles  in  rest,  one  fourth  to  the 
heart  and  the  large  blood-vessels,  one  fourth  to  the  liver,  and  one  fourth  to 
the  other  organs. 


Die  Blutvertheilung  und  der  Thatigkeitswechsel  der  Organe,  Leipzig,  1871. 


CHAPTER  VIl. 
CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

I.    Chyle  and  Lymph. 

The  lymph  is  the  mediator  in  the  exchange  of  constituents  between  the 
blood  and  the  tissues.  The  bodies  necessary  for  the  nutrition  of  the  tissues 
pass  from  the  blood  into  the  lymph,  and  the  tissues  deliver  water,  salts,  and 
products  of  metabolism  to  the  lymph.  The  lymph,  therefore,  originates 
partly  from  the  blood  and  partly  from  the  tissues.  From  a  purely  theo- 
retical standpoint  one  can,  according  to  Heidenhain,  differentiate  between 
blood-lymph  and  tissue-lymph  according  to  origin.  It  is  impossible  at  the 
present  time  to  completely  separate  that  which  comes  from  the  one  or  the 
other  source.  Chemically  the  lymph  is  the  same  as  plasma  and  contains,  at 
least  to  a  great  extent,  the  same  bodies.  The  observation  of  Asher  and 
Barbera,!  that  the  lymph  contains  poisonous  metabolic  products,  does 
not  contradict  such  an  assumption,  as  no  doubt  these  products  are  trans- 
ferred to  the  blood  with,  the  lymph.  Although  the  blood  does  not  show  the 
same  poisonous  action  as  the  lymph,  still  this  can  be  explained  by  the 
great  dilution  these  bodies  undergo  in  the  blood,  and  the  difference  between' 
blood-plasma  and  lymph  is  nojioubt  of  a  quantitative  nature.  This  differ- 
ence consists  chiefly  in  that  the  lymph  is  poorer  in  proteins.  No  essen- 
tial chemical  difference  has  been  found  between  the  lymph  and  the  chyle 
of  starving  animals.  After  fatty  food  the  chyle  differs  from  the  lymph  in 
its  wealth  of  minutely  divided  fat-globules,  which  give  it  a  milky  appear- 
a,nce;  hence  the  old  name  "lacteal  fluid." 

Chyle  and  lymph,  like  the  plasma,  contain  seralbimiin,  serglohulins, 
fibrinogen,  and  fibrin  ferment.  The  two  last-mentioned  bodies  occur  only 
in  very  small  amounts;  therefore  the  chyle  and  lymph  coagulate  slowly  (but 
spontaneously)  and  yield  but  little  fibrin.  Like  other  liquids  poor  in  fibrin 
ferment,  chyle  and  lymph  do  not  at  once  coagulate  completely,  but  repeated 
coagulations  take  place. 

The  extractive  bodies  seem  to  be  the  same  as  in  plasma.  Sugar  (or 
at  least  a  reducing  substance)  is  found  in  about  the  same  quantity  as  in  the 

'  Zeitschr.  f.  Biologic,  3(5. 

250 


CHYLE  AND  LYMPH.  251 

blood-serum,  but  in  larger  quantities  than  in  the  blood;  this  depends  on 
the  fact  that  the  blood-corpuscles  contain  no  sugar.  The  glycogen  detected 
by  Dastre  ^  m  the  lymph  occurs  only  in  the  leucocytes.  According  to 
RoHMANN  and  Bial/ lymph  contains  a  diastatic  enzyme  similar  to  that  in 
blood-plasma,  and  Lepixe  ^  has  found  that  the  chyle  of  a  dog  during 
digestion  has  great  glycolytic  activity.  The  amount  of  urea  has  been 
determined  by  Wurtz  ^  as  0.12-0.28  p.  m.  The  mineral  bodies  appear  to 
be  the  same  as  in  plasma. 

As  form-elements,  leucocytes  and  red  blood-corpuscles  are  common  to  both 
chyle  and  lymph.  Chyle  in  fasting  animals  has  the  appearance  of  lymph. 
After  fatty  food  it  is,  on  the  contrary,  milky,  due  partly  to  small  fat- 
globules,  as  in  milk,  and  partly,  indeed  mostly,  to  finely  divided  fat.  The 
nature  of  the  fat  occurring  in  chyle  depends  upon  the  kind  of  fat  in  the 
food.  By  far  the  greater  part  consists  of  neutral  fat,  and  even  after 
feeding  with  large  quantities  of  free  fatty  acids,  Munk*  found  that  the 
chyle  contained  chiefly  neutral  fat  with  only  small  amounts  of  fatty  acids 
or  soaps. 

The  gases  of  the  chyle  have  not  been  studied,  and  it  seems  that  the 
gases  of  an  entirely  normal  human  lymph  have  not  thus  far  been  investi- 
gated. The  gases  from  dog-lymph  contain  only  traces  of  oxygen  and 
consist  of  37.4-53.1  per  cent  CO2  and  1.6  per  cent  N,  calculated  at  0°  C, 
and  760  mm.  mercury.  The  chief  mass  of  the  carbon  dioxide  of  the  lymph 
seems  to  be  in  firm  chemical  combination.  Comparative  analyses  of  blood 
and  lymph  have  shown  that  the  lymph  contains  more  carbon  dioxide  than 
arterial,  but  less  than  venous  blood.  The  tension  of  the  carbon  dioxide 
of  lymph  is,  according  to  PFLtJGER  and  Strassburg,^  smaller  than  m 
venous,  but  greater  than  in  arterial  blood. 

The  quantitative  composition  of  the  chyle  must  e\idently  be  very  variable.^ 
The  analyses  thus  far  made  refer  only  to  that  mixtureof  chyle  and  lymph 
which  is  obtained  from  the  thoracic  duct.  The  specific  gravity  varies 
between  1.007  and  1.043.  As  an  example  of  the  composition  of  human  chyle 
two  analyses  will  be  given.  The  first  is  by  Owen-Rees,  of  the  chyle 
of  an  executed  person,  and  the  second  by  Hoppe-Seyler,'^  of  the  chyle  in 


'  Compt.  rend,  de  soc.  biol.,  47,  and  Compt.  rend.,  120;   Arch,  de  Physiol.  (5),  7. 
^  Rohmann  and  Bial,  Pfliiger's  .Arch.,  52,  53,  and  55;    L6pine,  Compt.  rend.,  110. 
^  Compt.  rend.,  49. 

"  Virchow's  Arch.,  SO  and  123.     In  regard  to  the  analysis  of  the  fat  of  chyle,  see 
Erben,  Zeitschr.  f.  physiol.  Chem.,  30. 

*  Hanamarsten,  Die  Gase  der  Hundelymphe,  Arbeiten  aus  d.  physiol.  Anstalt  zu 
Leipzig,  1871;    Strasburg,  Pfliiger's  Archiv,  6. 

•  See  also  Panzer,  Zeitschr.  f.  physiol.  Chem.,  30. 

'  Owen-Rees,  cited  from  Hoppe-Seyler's  Physiol.  Chem.,  595;   Hoppe-Seyler,  ibid., 
.597.     See  also  Carlier,  Brit.  Med.  Journ.,  1902,  175. 


252  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

a  case  of  rupture  of  the  thoracic  duct.     In  the  latter  case  the  fibrin  had 
previously  separated.     The  results  are  in  1000  parts. 

No.  1.  No.  2. 

Water 904.8         940.72  water 

Solids 95 . 2  59 .  28  solids 

Fibrin Traces  

Albumin 70.8  36.67  albumin 

Fat 9.2  7.23  fat 

f  2 .  35  soaps 
i  0 .  83  lecithin 
Remaining  organic  bodies  ...     10.8         -j  1.32  cholesterin 

3 .  63  alcohol  extractives 
[  0 .  58  water  extractives 
Q„, .  _  ^    .  /  6 .  80  soluble  salts 

'^^"^ : ^-^         \  0.35  insoluble  salts 

The  quantity  of  fat  is  very  variable  and  may  be  considerably  increased 
by  partaking  of  food  rich  in  fats.  I.  Muxk  and  A.  Rosenstein  ^  have  inves- 
tigated the  lymph  or  chyle  obtained  from  a  lymph  fistula  at  the  end  of  the 
upper  third  of  the  leg  of  a  girl  eighteen  years  old  and  weighing  60  kg.,  and 
the  highest  quantity  of  fat  in  the  chylous  lymph  was  47  p.  m.  after  par- 
taking of  fat.  In  the  starvation  lymph  from  the  same  patient  they  found 
only  0.6-2.6  p.  m.  fat.  The  quantity  of  soaps  was  always  small,  and  on  par- 
taking of  41  grams  of  fat  the  quantity  of  soaps  was  only  about  ^o  of  the 
neutral  fats. 

A  great  many  analyses  of  chyle  from  animals  have  been  made,  and 
they  chiefly  show  the  fact  that  the  chyle  is  a  liquid  with  a  very  changeable 
composition  which  stands  closely  related  to  blood-plasma,  but  with  the 
chief  difference  that  it  contains  more  fat  and  less  solids.  The  reader  is 
referred  to  special  works  for  these  analyses,  as,  for  example,  to  v.  Gorup- 
Besanez's  "Lehrbuch  der  physiologischen  Chemie,"  4th  edition. 

The  composition  of  the  lymph  is  also  very  changeable,  and  its  specific 
gravity  shows  about  the  same  variation  as  the  chyle.  In  the  following 
analyses,  1  and  2,  made  by  Gubler  and  Quevenne,  are  the  results  ob- 
tained from  lymph  from  the  upper  part  of  the  thigh  of  a  woman  aged 
thirty -nine;  and  3,  made  by  v.  Scherer,  is  an  analysis  of  lymph  from 
the  sac-like  dilated  lymphatic  vessels  of  the  spermatic  cord.  No.  4  was 
made  by  C.  Schmidt,^  the  data  being  obtained  from  lymph  from  the  neck 
of  a  colt.    The  results  are  expressed  in  parts  per  1000. 

12  3  4 

Water 939.9  934.8  957.6  955.4 

Solids 60.1  65.2  42.4  44.6 

Fibrin 0.5  0.6  0.4  2.2 

Albumin 42.7  42.8  34.7]  .... 

Fat,  chole.sterin,  lecitliin 3.8  9.2  I  35 . 0 

Extractive  bodies 5.7  4.4  ■••]  .... 

Salts 7.3  8.2  7.2  7.5 

'  Virchow's  Arch.,  123. 

*  Gubler  and  Quevenne,  cited  from  Hoppe-Seyler's  Physiol.  Chem.,  591;  v.  Scherer^ 
ibid.,  591;  C.  Schmidt,  ibid.,  592. 


LYMPH.  253 

The  salts  found  by  C.  Schmidt  in  the  lymph  of  the  horse  have  the  fol- 
lowing composition,  calculated  in  parts  per  1000  parts  of  the  lymph: 

Sodium  chloride 5 .  67 

Soda 1 .27 

Potash 0. 16 

Sulphuric  acid 0 .  09 

Phosphoric  acid  united  with  alkalies 0.02 

Earthy  phosphates 0 .  26 

In  the  cases  investigated  by  ^Iuxk  and  Rosenstein  the  quantity  of 
solids  in  the  fasting  condition  varied  between  35.7  and  57.2  p.  m.  This 
variation  was  essentially  dependent  upon  the  extent  of  secretion,  so  that 
the  low  amount  coincides  with  a  more  active  secretion,  and  the  reverse  in 
the  other  case.  The  chief  portion  of  the  solids  consisted  of  proteins,  and 
the  relationship  between  globulin  and  albumin  was  as  1:2.4  to  4.  The 
mineral  bodies  in  1000  parts  lymph  (chylous)  were:  NaCl  5.83;  Na2C03  2.17; 
K2HPO4  0.28;   Ca3(P04)2  0.28;   :\Ig3(P04)2  0.09;   and  Fe(P04)  0.025. 

Under  special  conditions  the  lymph  may  be  so  rich  in  finely  divided  fat 
that  it  appears  like  chyle.  Such  lymph  has  been  investigated  by  Hexsen 
in  a  case  of  lymph  fistula  in  a  ten -year-old  boy,  and  by  Laxg  ^  in  a  case  of 
lymph  fistula  in  the  upper  part  of  the  left  thigh  of  a  girl  of  seventeen. 
The  lymph  investigated  by  Hexsex  varied  in  the  quantity  of  fat,  as  an 
average  of  nineteen  anah'ses,  between  2.8  and  36.9  p.  m.,  while  that  inves- 
tigated by  Laxg  contained  24.85  p.  m.  of  fat. 

The  quantity  of  lymph  secreted  must  naturally  change  considerably 
under  various  conditions,  and  there  are  no  means  of  measuring  it.  The 
size  of  the  flow  of  lymph  is,  as  Heidenhain  suggests,  no  measure  of  the 
abundance  of  supply  of  nutritive  material  to  the  organs,  and  the  lymph- 
tubes  act  according  to  him  as  "drain-tubes,"  remo\ang  the  excess  of  fluid 
from  the  lymph-fissures  as  soon  as  the  pressure  therein  rises  to  a  certain 
height.  Attempts  have  been  made  to  determine  the  quantity  of  lymph 
flowing  in  24  hours  in  the  thoracic  duct  of  animals.  According  to 
Heidexhaix  the  quantity  averages  640  c.c.  for  a  dog  weighing  10  kilos. 

Determinations  of  the  quantity  of  lymph  in  man  have  also  been 
attempted.  Noel-Patox^  obtained  1  c.c.  of  lymph  per  minute  from  the 
severed  thoracic  duct  of  a  patient  weighing  60  kilos.  The  quantity  in  the 
24  hours  cannot  be  calculated  from  this  amount.  In  the  case  of  ]Muxk  and 
Rosexsteix,  1134-1372  grams  chyle  was  collected  -uithin  12-13  hours  after 
partaking  of  food.  In  the  fasting  condition  or  after  starving  for  18  hours 
they  found  50  to  70  grams  per  hour,  sometimes  120  grams  and  above,  espe- 
cially in  the  first  few  hours  after  powerful  muscular  exercise. 

Several  circumstances  have  a  marked  influence  on  the  extent  of  lymph 

'  Hensen,  Pfliiger's  Arch.,  10;   Lang,  see  Maly's  Jahresber.,  4. 
^  Journ.  of  Physiol.,  11. 


254     CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

secretion.  During  starvation  less  lymph  is  secreted  than  after  partaking 
of  food.  Nasse  1  has  observed  in  dogs  that  the  formation  of  lymph  is 
increased  36  per  cent  more  after  feeding  with  meat  than  after  feeding  with 
potatoes,  and  about  54  per  cent  more  than  after  24  hours'  deprivation  of 
food.  In  this  connection  mention  must  be  made  of  the  important  observa- 
tions of  AsHER  and  Barbera  ~  that  with  pure  protem  diet  the  lymph 
current  is  increased  in  the  thoracic  ca\'ity,  and  also  that  the  increase  in  the 
lymph  secretion  runs  parallel  with  the  elimination  of  nitrogen  in  the  urine, 
i.e.,  with  the  absorption  of  the  protein  from  the  digestive  tract. 

An  increase  in  the  total  quantity  of  blood,  as  by  transfusion  of  blood, 
also  especially  on  preventing  the  flow  of  blood  by  means  of  ligatures,  causes 
an  increase  in  the  quantity  of  lymph.  According  to  Heidenhain,  on  the 
contrary,  a  very  considerable  change  in  the  pressure  in  the  aorta  causes 
only  a  little  change  in  the  abundance  of  the  lymph-flow.  The  quantity  of 
lymph  may  be  raised  by  powerfully  active  and  passive  movements  of  the 
limbs  (Lesser).  Under  the  influence  of  curare,  an  increase  of  the  lymph 
secretion  is  observed  (Paschutin,  Lesser^),  and  the  quantity  of  solids  in 
the  lymph  is  also  increased. 

The  bodies  inciting  lymph-flow,  the  so-called  himphagogues,  are  of  espe- 
cially great  interest,  and  they  may,  accordmg  to  Heidenhain,^  be  divided 
into  two  different  chief  groups.  The  lymphagogues  of  the  first  series — 
extracts  of  crab-muscles,  blood-leech,  anodons,  liver  and  intestme  of  dogs, 
as  well  as  peptone  and  egg  albumin,  strawberry  extracts,  metabolic  products 
of  bacteria  and  others — cause  a  greatly  increased  secretion  of  lymph  with- 
out raising  the  blood-pressure,  and  in  tliis  way  the  blood-plasma  becomes 
poorer  in  proteins  and  the  lymph  richer  than  before.  For  the  formation 
of  this  lymph,  which  Heidenhain  designates  blood-lymph,  we  must  admit 
with  him  that  a  special  secretory  activity  of  the  capillary-wall  endothelium 
exists.  The  lymphagogues  of  the  second  series,  such  as  sugar,  urea,  sodium 
chloride,  and  other  salts,  also  cause  an  abundant  lymph  formation.  The 
blood,  as  well  as  the  lymph,  thereby  becomes  richer  in  w'ater.  This 
increased  amount  of  water  depends,  according  to  Heidenhain,  upon  an  in- 
creased delivery  of  water  by  the  tissue-elements,  and  this  lymph  is  chiefly 
tissue-lymph,  according  to  him.  Diffusion  is  no  doubt  of  great  impor- 
tance in  the  formation  of  this  lymph,  but  the  secretory  acti\ity  of  the 
endothelium  is  also  of  importance,  at  least  for  certain  bodies,  such  as  sugar. 

'  Cited  from  Hoppe-Seyler,  Physiol.  Chem.,  593. 

'  The  works  of  Asher  and  collaborators,  Barbera,  Gies,  and  Busch,  upon  lymph 
formation  may  be  found  in  Zeitschr.  f.  Biologie,  3(5,  37,  40. 

^  Lesser,  Arbeiten  aus  der  physiol.  Anstalt  zu  Leipzig,  Jahrgang  6;  Paschutin, 
ibid.,  7. 

^  Heidenhain,  Pfiiiger's  Arch.,  49;  Hamburger,  Zeitschr.  f.  Biologie,  27  and  30. 
See  especially  Ziegler's  Beitr.  zur  Path.  u.  zur  allg.  Pathol.,  14,  443;  also  Arch.  f. 
(Anat.  u.)  Physiol.,  189.5  and  1896. 


LYMPH  FORMATION.  255 

In  the  past,  the  formation  of  lymph  was  explained  in  a  purely  physical 
way  by  the  united  action  of  filtration  from  the  blood  and  the  osmosis 
between  the  blood  and  tissue-fluid.  Later  Heidenhain  and  Hamburger 
ascribed  a  special  activity  to  the  capillar}^  endothelium,  assuming  that 
they  take  part  m  the  formation  of  lymph  in  a  secretory'  manner. 

Another  view  which  also  besides  the  physical  processes  is  of  especial 
physiological  moment  in  the  explanation  of  lymph  formation  was  sug- 
gested by  AsHER  and  his  collaborators  (Barbera,  Gies,  and  Busch), 
According  to  them  the  lymph  is  a  product  of  the  work  of  the  organs;  it& 
amount  is  dependent  upon  an  increased  or  diminished  activity  of  the  organs,, 
and  the  lymph  is  therefore  a  measure  of  the  work  in  these.  The  close 
relation  between  lymph  formation  and  the  work  of  organs  has  also  been 
shown  for  several  of  them,  especially  for  the  liver.  Starling  has  shown 
that  after  the  introduction  of  lymphagogues  of  the  first  series,  chiefly  liver 
lymph  is  secreted,  which  he  claims  is  a  proof  against  Heidenhain's  \dew^ 
and  he  explains  the  increased  permeability  of  the  vessel  wall  by  the  fact  that 
these  bodies  have  a  poisonous  irritating  action.  On  the  contran,-,  Ashek 
explains  this  increased  lymph-flow  by  the  statement  that  the  substance  in 
question — as  well  as  those  influences  which  incite  the  acti\'ity  of  the  liver 
— produces  an  increased  formation  of  lymph  in  these  organs.  This  view  is 
supported  by  experiments  upon  the  action  of  lymphagogues  on  blood  coag- 
ulation and  liver  activity  (Delezenne  and  others),  for,  according  to  Gley, 
these  bodies  have  at  the  same  time  a  lymphagogue  action  and  an  action 
upon  the  secretion  of  the  glands.  We  have  no  direct  evidence  of  the  action 
of  the  lymphagogues  of  the  first  series  upon  the  organs,  but  we  know  from 
Kusmine's  work  that  peptone,  leech  extract,  and  the  extractives  of  the 
crab-muscles  act  directly  upon  the  liver-cells  and  bring  about  morpho- 
logical changes.  The  connection  between  organ  activity  and  lymph  for- 
mation has  also  been  sho\\ai  upon  muscles  and  glands  by  others  besides  the 
above-mentioned  investigators  (Hamburger,  Bainbridge  i). 

The  extent  of  organ  work  certainly  essentially  influences  the  quantity 
and  properties  of  the  lymph.  Still  from  this  we  cannot  draw  any  positive 
conclusions  as  to  whether  the  lymph  formation  is  brought  about  by  physico- 
chemical  processes  alone  or  whether  in  this  process  a  specific,  not  closely 
definable  secretory  force  is  at  work  at  the  same  time.  In  regard  to  this 
much-disputed  question  attention  must  be  called  in  the  first  place  to  the 
fact  that  the  important  works  of  Heidenhain,  Hamburger,  Lazarus- 
Barlow,  and  others,  as  well  as  the  investigations  of  Asher  and  Gies  and  of 
Mendel  and  Hooker  ^  upon  the  lengthy  post-mortem  lymph-flow,  have 

'  In  regard  to  the  works  cited,  as  well  as  the  literature  upon  lymph  formation,  see 
ElHnger,  "Die  Bildung  der  Lymphe,"  Ergebnisse  der  Physiol.,  I,  Abt.  1,  1902,  and 
Asher,  Biochem.  Centralbl.,  4. 

^  Anier.  Journ.  of  Physiol.,  7. 


256  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

shoT\Ti  that  the  older  filtration  hypothesis  is  untenable.  That  the  part 
played  by  filtration  as  compared  with  that  of  the  osmotic  force  is  only 
very  trivial  has  been  conclusively  shown  by  the  adherents  of  the  physico- 
chemical  theory  of  lymph  formation. 

Several  investigators  (Koranyi,  Starling,  Roth,  Asher,  and  others) 
have  shown  clearly  that  the  work  in  the  glands  and  tissue-cells  must  cause  a 
difference  in  the  osmotic  pressure  upon  the  two  sides  of  the  capillary  wall. 
That  this  is  so  follows  from  several  circumstances  and  especially  from  the 
fact  that,  in  dissimilation  in  the  cells,  bodies  of  high  molecular  weight  are 
split  into  a  number  of  smaller  molecules,  which  latter,  either  directly,  if  they 
leave  the  cells  and  pass  into  the  tissue-fluid,  or  indirectly,  when  they  remain 
in  the  cells,  produce  an  increase  in  the  osmotic  tension  within  the  cells,  and 
in  this  way  cause  a  taking  up  of  water  from  the  fluid  and  must  therefore 
increase  the  osmotic  pressure  of  the  tissue-fluids.  As  the  cells  can  by  syn- 
thesis build  up  highly  complex  constituents  from  simple  molecules,  and  as 
the  chief  products  of  catabolism  are  carbon  dioxide  and  water,  it  is  diffi- 
cult to  explain  these  intricate  conditions.  Still,  irrespective  of  whatever 
\aew,  a  change  in  one  or  the  other  direction  in  the  osmotic  pressure  upon 
both  sides  of  the  capillary  wall  must  be  produced  hereby.  Whether  this 
and  other  physico-chemical  processes  are  alone  sufficient  to  explain  the 
lymph  formation  (Cohnstein,  Ellinger)  remains  an  open  and  disputed 
question.* 

II.    Transudates  and  Exudates. 

The  serous  membranes  are  normally  kept  moistened  by  liquids  whose 
quantity  is  sufficient  only  in  a  few  instances,  as  in  the  pericardial  cavity 
and  the  subarachnoidal  space,  for  a  complete  chemical  analysis  to  be  made 
of  them.  Under  diseased  conditions  an  abundant  transudation  may  take 
place  from  the  blood  into  the  serous  cavities,  into  the  subcutaneoJis  tissues, 
or  under  the  epidermis;  and  in  this  way  pathological  transudates  are 
formed.  Such  tme  transudates,  which  are  similar  to  lymph,  are  gener- 
ally poor  in  form-elements  and  leucocytes,  and  yield  only  very  little  or 
almost  no  fibrin,  while  the  inflammatory  transudates,  the  so-called  exu- 
dates, are  generally  rich  in  leucocytes  and  yield  proportionally  more  fibrin. 
As  a  rule,  the  richer  a  transudate  is  in  leucocytes  the  closer  it  stands  to 
pus,  while  a  diminished  quantity  of  leucocytes  renders  it  more  nearly  like  a 
real  transudate  or  lymph. 

It  is  ordinarily  accepted  that  filtration  is  of  the  greatest  importance 
in  the  formation  of  transudates  and  exudates.  The  facts  coincide  with 
this  \aew  that  all   these  fluids  contain  the  salts  and  extractive  bodies 

'  On  this  question  see  Ellinger,  "Die  Bildung  der  Lymphe,"  Ergebnisse  der  Phy- 
eiologie,  I,  Abt.  1,  355,  and  Asher,  Biochem.  Centralbl.,  4,  pp.  1  and  45. 


TRANSUDATES  AND  EXUDATES.  257 

occurring  in  the  blood-plasma  in  about  the  same  quantity  as  the  blood- 
plasma,  while  the  amount  of  proteins  is  habitually  smaller.  T\Tiile  the 
different  fluids  belonging  to  this  group  have  about  the  same  quantities  of 
salts  and  extractive  bodies,  they  differ  from  one  another  chiefly  in  con- 
taining differing  quantities  of  protein  and  form-elements,  as  well  as  vary- 
ing quantities  of  transformation  and  decomposition  products  of  these 
latter — changed  blood-coloring  matters,  cholesterin,  etc.  The  correspond- 
ence in  the  amount  of  salts  and  extractive  bodies  present  in  the  blood  and 
in  transudates  supplies  just  as  little  proof  for  a  filtration  as  it  does  for 
the  formation  of  lymph;  but  still  it  cannot  be  doubted  for  other  reasons 
that  filtration  is  often  of  great  importance  in  the  formation  of  a  transu- 
date. To  what  extent  filtration  is  active  in  the  perfectly  normal  vascular 
wall  cannot  be  answered. 

The  altered  permeability  of  the  capillary-  walls  in  disease  is  a  second 
important  factor  in  the  formation  of  transudates.  The  circumstance  that 
the  greatest  quantity  of  protein  occurs  in  transudates  in  inflammatory 
processes,  to  which  is  also  due  the  abundant  quantity  of  form-elements  in 
such  transudates,  has  been  explained  by  this  hypothesis.  The  greater 
quantity  of  protein  in  the  transudates  in  formative  irritation  is  in  great 
part  explained  by  the  large  amount  of  destroyed  form-elements.  The 
interesting  observation  made  by  Paijkull,^  that  in  such  cases  in  which  an 
inflammator}-  irritation  has  taken  place  the  fluid  contains  nucleoalbumin 
(or  nucleoproteid?),  while  this  substance  does  not  occur  in  transudates 
in  the  absence  of  inflammatory'  processes,  can  be  explained  by  the  pres- 
ence of  form-elements.  Still,  such  a  phosphorized  protein  substance  does 
not  occur  in  all  inflammatory^  exudates. 

As  the  secretor}^  importance  of  the  capillar}^  endothelium  has  been  made 
probable  by  the  investigations  of  Heidexhaix,  it  is  a  priori  to  be  expected 
that  an  abnormallv  increased  secretory-  acti\'ity  of  the  endothelium  is  a 
cause  of  transudates.  Those  observations  which  substantiate  such  an 
assumption  can  also  be  explained  just  as  well  by  assuming  a  changed 
permeability  of  the  capillar}-  walls. 

The  var}-ing  quantities  of  protein  observed  by  C.  Schacdt^  in  the 
tissue-fluids  in  different  vascular  regions  can  perhaps  be  explained  by  the 
different  condition  of  the  capillarv  endothelium.  For  example,  the  amount 
of  protein  in  the  pericardial,  pleural,  and  peritoneal  fluids  is  con- 
siderably greater  than  in  those  fluids  which  are  found  in  the  subarach- 
noidal space,  in  the  subcutaxeous  tissues,  or  in  the  aqueous  humor, 
which  are  poor  in  protein.  The  condition  of  the  blood  also  greatly  affects 
the  transudates,  for  in  hydrsemia  the.  amount  of  protein  in  the  transudate 

•  See  Maly's  Jahresber.,  22. 

'  Cited  from  Hoppe-Seyler,  Physiol.  Chem.,  607 


258  CHYLE,  LY^IPH,  TRANSUDATES  AND  EXUDATES. 

is  very  small.  With  the  increase  in  the  age  of  a  transudate,  of  a  hydrocele 
fluid  for  instance,  the  quantity  of  protein  is  increased,  probably  by  resorp- 
tion of  water,  and  indeed  exceptional  cases  may  occur  in  which  the  amount 
of  protein,  without  any  previous  hemorrhage,  is  even  greater  than  in  the 
blood-serum. 

The  proteins  of  transudates  are  chiefly  seralbumin,  serglobulin,  and  a 
little  fibrinogen.  Proteoses  and  peptones  do  not  occur,  excepting  perhaps 
in  the  cerebrospinal  fluid,  and  in  those  cases  where  an  autolysis  has  taken 
place  in  the  liquid.^  The  non-inflammatory  transudates  as  a  rule  undergo 
spontaneous  coagulation  not  at  all,  or  only  very  slowly.  On  the  addition  of 
blood  or  blood-serum  they  coagulate.  Inflammatory  exudates  coagulate 
spontaneously,  and  Paijkull  has  shown  that  these  often  contain  nucleo- 
proteid  (or  nucleoalbumin).  In  inflammatory'  exudates  a  protein  sub- 
stance has  been  habitually  observed  which  is  precipitated  by  acetic  acid, 
but  which  does  not  occur  in  transudates,  or  only  in  very  small  quantities. 
This  substance,  which  has  been  observed  and  studied  by  ]\Ioritz,  Staehelin, 
Umber,  and  Rivalta,  is  claimed  by  the  first  three  observers  to  be  free 
from  phosphorus,  while  Rivalta  considers  it  to  be  a  phosphorized  pseudo- 
globulin.  Umber  calls  it  serosamucin,  although  it  yields  only  very  little 
reducing  carbohydrate.  According  to  Joachim  ^  it  is  only  a  part  of  the 
globulin,  a  view  which  cannot  be  correct  for  all  cases,  v.  Holst^  has  so 
far  substantiated  Umber's  observation  in  that  he  has  isolated  a  mucin 
substance  from  an  ascitic  fluid  in  carcinoma  of  the  stomach  and  the  peri- 
toneum, which  seemed  to  be  identical  with  Umber's  serosamucin,  as  well 
as  with  the  synovial  mucin.  There  does  not  seem  to  be  any  doubt  that 
in  transudates  and  exudates  different  protein  substances  may  occur  under 
different  circumstances,  although  the  globulins  form  besides  seralbumin 
ordinarily  the  chief  mass  of  the  protein  bodies.  ^Mucoid  substances,  which 
were  first  observed  by  Hammarsten  in  certain  cases  of  ascites  without 
complications  with  ovarial  tumors,  and  which  are  cleavage  products  of  a 
more  complicated  substance,  seem  according  to  Paijkull  "*  to  be  regular 
constituents  of  transudates  and  are  closely  related  to  the  above-mentioned 
serosamucin. 

There  are  numerous  investigations  on  the  relationship  between  globu- 
lin and  seralbumin,  and  Joachim  has  recently  determined  the  relationship 

'  Umber,  Miinch.  med.  Wochenschr.,  1902,  and  Berlin,  klin.  Wochenschr.,  1903. 
In  regard  to  the  autolysis  in  transudates,  see  also  Galdi,  Biochem.  Centralbl.,  3;  Ep- 
pinger,  Zeitschr.  f.  Heilkunde,  3.");  and  Zak,  Wien.  klin.  Wochenschr.,  1905. 

'Paijkull,  1.  c;  Moritz,  Miinch.  med.  Wochenschr.,  1903;  Staehelin,  ibid.,  1902; 
Umber,  Zeitschr.  f.  klin.  Med.,  48;  Rivalta,  Biochem.  Centralbl.,  2  and  5;  Joachim, 
Pfliiger's  Arch.,  93. 

^  Zeitschr.  f.  physiol.  Chem.,  43. 

*  Hammarsten,  ibid.,  15;   Paijkull,  1.  c. 


TRANSUDATES  AND   EXUDATES.  259 

between  euglobulin  and  the  total  globulin.  No  conclusive  results  can  be 
drawn  from  these  determinations.  The  relationship  between  globulin  and 
seralbumin  varies  very  much  in  different  cases,  but,  as  Hoffmann  and 
PiGEAND  ^  have  shown,  the  variation  is  in  each  case  the  same  as  in  the 
blood-serum  of  the  indi\ddual. 

The  specific  gravity  runs  nearly  parallel  with  the  quantity  of  protein. 
The  varying  specific  gravity  has  been  suggested  as  a  means  of  differentiation 
between  transudates  and  exudates  by  Reuss,^  as  the  first  oftdn  show  a 
specific  gravity  below  1015-1010,  while  the  others  have  a  specific  gra\'ity 
of  1018  or  above.    This  rule  holds  good  in  many,  but  not  in  all  cases. 

The  gases  of  the  transudates  consist  of  carbon  dioxide  besides  small 
amounts  of  nitrogen  and  traces  of  oxygen.  The  tension  of  the  carbon 
dioxide  is  greater  in  the  transudates  than  in  the  blood.  When  mixed 
with  pus,  the  amount  of  carbon  dioxide  is  decreased. 

The  extractives  are,  as  above  stated,  the  same  as  in  the  blood-plasma; 
but  sometimes  extractive  bodies  occur,  such  as  allantoin  in  dropsical  fluids 
(MoscATELLi^),  which  "lave  not  been  detected  in  the  blood.  Urea  seems 
-to  occur  in  vexy  variable  amounts.  Sugar  also  occurs  in  transudates,  but 
it  is  not  known  to  what  extent  the  reducing  power  is  due,  as  in  blood- 
serum,  to  other  bodies.  A  reducing,  non-fermentable  substance  has  been 
found  by  Picil^rdt  in  transudates.  The  sugar  is  generally  dextrose,  but 
le\'ulose  seems  to  have  been  found*  in  several  cases.  Sarcolactic  acid  has 
been  found  by  C.  Kulz  ^  in  the  pericardial  fluid  from  oxen.  Succinic  acid 
has  been  found  in  a  few  cases  in  hydrocele  fluids,  while  in  other  cases  it 
is  entirely  absent.  Leucine  and  tyrosine  have  been  found  in  transudates 
from  diseased  livers  and  in  pus-like  transudates  which  have  undergone 
decomposition,  and  after  autolysis.  Among  other  extractives  found  in 
transudates  must  be  mentioned  uric  acid,  xanthine,  creatine,  inosite,  and 
jn/rocatechin  (?). 

The  di\isio"n  of  the  nitrogenous  substances  in  human  transudates  and 
exudates  has  so  far  been  little  studied.  Otori  ^  has  found  that  no  essential 
difference  exists  between  serous  exudates  and  transudates  in  regard  to  the 
quantity  of  urea  and  amino-acids.  The  amount  of  total  nitrogen  and 
proteins  runs  parallel  with  the  specific  gravity,  and  the  same  is  generally 
true  for  the  absolute  values  for  amino-acid  nitrogen  and  purine  nitrogen. 

'  Joachim,  I.  c;  Hoffmann,  Arch.  f.  exp.  Path.  u.  Pharra.,  10;  Pigeand,  see  Maly's 
Jahresber.,  16. 

Reuss,  Deutsch.  Arch.  f.  klin.  Med.,  28.     See  also  Otto,  Zeitschr.  f.  Heilkunde,  17. 

'  Zeitschr.  f.  physiol.  Chem.,  13. 

*  Pickardt,  Berl.  klin.  Wochenschr.,  1897.  See  also  Rotmann,  Miinch.  med.  Woch- 
enschr.,  1898;   Neuberg  and  Strauss,  Zeitschr.  f.  physiol.  Chem.,  36. 

»  Zeitschr.  f.  Biologic,  32. 

'  Zeitschr.  f.  Heilkunde,  25. 


260  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

The  aniino-acid  nitrogen  and  the  urea  nitrogen  in  pus  are  greater  as  the 
specific  gravity  rises.  In  serous  exudates  and  transudates,  on  the  contrary, 
the  amino-acid  nitrogen  and  the  urea  nitrogen  are  not  proportional  to  the 
specific  gravity,  but  are  dependent  upon  the  general  circulator}'  condition 
of  the  body. 

The  investigations  upon  the  molecular  concentration  have  shown  that 
no  essential  and  constant  difference  exists  between  exudates  and  transu- 
dates. The  osmotic  concentration  and  the  concentration  of  the  electrolytes 
are  as  a  rule  the  same  as  in  blood-serum,  although  sometimes  rather  di- 
vergent results  have  been  found.  The  concentration  of  the  electrolytes 
shows  according  to  Bodon,^  like  the  blood-serum,  much  less  variation  than 
the  total  concentration.  The  alkalinity  determined  by  titration  is  about 
the  same  in  transudates  and  exudates  and  is  equal  to  that  of  the  blood- 
serum.  The  determination  of  the  HO-ion  concentration  has  shown  that  the 
transudates  and  exudates  in  this  regard  are  about  as  neutral  as  the  blood- 
serum  (Bodon). 

As  above  stated,  irrespective  of  the  varying  number  of  form-elements 
contained  in  the  different  transudates,  the  quantity  of  protein  is  the  most 
characteristic  chemical  distinction  in  the  composition  of  the  various  trans- 
udates; therefore  a  quantitative  analysis  is  of  importance  only  in  so  far 
as  it  considers  the  quantity  of  protein.  On  this  account,  in  the  following, 
relative  to  the  quantitative  composition,  chief  stress  will  be  put  on  the 
quantity  of  protein. 

Pericardial  Fluid.  The  quantity  of  this  fluid  is,  even  under  physio- 
logical conditions,  so  large  that  a  sufficient  quantity  for  chemical  inves- 
tigation has  been  obtained  (from  persons  who  had  been  executed).  This 
fluid  is  lemon-yellow  in  color,  somewhat  sticky,  and  yields  more  fibrin  than 
other  transudates.  The  amount  of  solids,  according  to  the  anal3'ses  per- 
formed by  v.  Gorup-Besanez,  Wachsmuth,  and  Hoppe-Seyler,^  is 
37.5-44.9  p.  m.,  and  the  amount  of  protein  is  22.8-24.7  p.  m.'  The  anal3^sis 
made  by  Hammarsten  of  a  fresh  pericardial  fluid  from  a  young  man  who 
had  been  executed  yielded  the  following  results,  calculated  in  1000  parts  by 
weight: 

Water 960.85 

Solids 39.15 

f  Fibrin 0.31 

Proteins 28. 60 ]  Globulin 5.95 

I  Albumin  ...   22 .  34 

Solublesalts 8.60     NaCl 7.28 

Insoluble  salts 0.15 

Extractive  bodies 2 .  00 


'  Pfliiger's  Arch.,  104,  where  the  literature  on  this  subject  may  be  found. 
^  V.  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4.  Aufl.,401;  Wachsmuth,  Vir- 
chow's  Arch.,  7;   Hoppe-Seyler,  Physiol.  Chem.,  605. 


PLEUR.IL  FLUID.  261 

Friend  ^  has  found  nearly  the  same  composition  for  a  pericardial  fluid 
from  a  horse,  -^ith  the  exception  that  this  liquid  was  relatively  richer  in 
globulin.  The  ordinary-  statement  that  pericardial  fluids  are  richer  in 
fibrinogen  than  other  transudates  is  hardly  based  on  sufficient  proof.  In 
a  case  of  chylopericardium,  which  was  probably  due  to  the  rupture  of  a 
chylous  vessel  or  caused  by  a  capillary  exudation  of  chyle  because  of  stop- 
page, Hasebroek^  found  in  1000  parts  of  the  fluid  103.61  parts  solids, 
73.79  parts  proteins,  10.77  parts  fat,  3.34  parts  cholesterin,  1.77  parts 
lecitliin,  and  9.34  parts  salts. 

The  pleural  fluid  occurs  under  physiological  conditions  in  such  small 
quantities  that  a  chemical  analysis  of  it  cannot  be  made.  Under  patho- 
logical conditions  this  fluid  may  show  verj^  variable  properties.  In  cer- 
tain cases  it  is  nearly  serous,  in  others  again  sero-fibrinous,  and  in  others 
similar  to  pus.  There  is  a  corresponding  variation  in  the  specific  gra\'ity 
and  the  properties  in  general.  If  a  pus-like  exudate  is  kept  enclosed  for  a 
long  time  in  the  pleural  ca\dty,  a  more  or  less  complete  maceration  and 
solution  of  the  pus-corpuscles  is  found  to  take  place.  The  ejected  3-elloT\ish- 
brown  or  greenish  fluid  may  then  be  as  rich  in  solids  as  the  blood-serum; 
and  an  abundant  fiocculent  precipitate  of  a  nucleoalbumin  or  nucleopro- 
teid  (the  pyin  of  early  -^Titers)  may  be  obtained  on  the  addition  of  acetic 
acid.     This  precipitate  is  soluble  with  difficulty  in  an  excess  of  acetic  acid. 

Numerous  analyses,  b}'  many  investigators .^  of  the  quantitative  com- 
position of  pleural  fluids  under  pathological  conditions  have  teen  published. 
From  these  analyses  we  learn  that  in  hydrot  borax  the  specific  gra\dty  is 
lower  and  the  quantity  of  protein  less  than  in  pleuritis.  In  the  first  case 
the  specific  gra\-ity  is  generally  less  than  1.015,  and  the  quantity  of  protein 
10-30  p.  m.  In  acute  pleuritis  the  specific  g^a^'ity  is  generally  higher  than 
1020,  and  the  c[uantity  of  protein  30-65  p.  m.  The  quantity  of  fibrinogen, 
which  in  hydrothorax  is  about  0.1  p.  m.,  may  amount  to  more  than  1  p.  m. 
in  pleuritis.  In  pleurisy  wth  an  abundant  accumulation  of  pus,  the  specific 
gra\'ity  may  rise  even  to  1.030,  according  to  the  observations  of  H.^enl^r- 
STEN.  The  quantity  of  solids  is  often  60-70  p.  m..  and  may  be  even  more 
than  90-100  p.  m.  (HA^LMARSTEx).  ^lucoid  substances  have  also  been 
detected  in  pleural  fluids  by  Paijkull.  Cases  of  chylous  pleurisy  are  also 
known;  in  such  a  case  ^Iehu*  found  17.93  p.  m.  fat  and  cholesterin  in 
the  fluid. 

The  quantity  of  peritoneal  fluid  is  very  small  under  physiological  condi- 

*  Halliburton,  Text-book  of  Chem.  Physiol,  etc.,  London,  1904. 

-  Zeitschr.  f.  physiol.  Chem.,  12. 

'  See  the  works  of  Mehu,  Runeberg,  F.  HofTmann,  Reuss,  all  of  which  are  cited  in 
Bemheim's  paper  in  Virchow's  Arch.,  131,  274.  See  also  Paijkull  1  c,  and  Halli- 
burton's Text-book,  .346;   Joachim,  1.  c. 

*Arch.  gen.  de  med.,  1886,  2,  cited  from  Maly's  Jahresber..  16. 


262  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

tions.  The  investigations  refer  only  to  the  fluid  under  diseased  conditions 
(dropsical  or  ascitic  fluid).  The  color,  transparency,  and  consistency  of 
these  may  vary  greatly. 

In  cachectic  conditions  or  a  hydrgemic  condition  of  the  blood  the  fluid 
has  little  color,  is  milky,  opalescent,  watery,  does  not  coagulate  spon- 
taneously, has  a  very  low  specific  gravity,  1.005-1.010-L015.  and  is  nearly 
free  from  form-elements.  The  ascitic  fluid  in  portal  stagnation,  or  in 
general  venous  congestion,  has  a  low  specific  gravity  and  ordinarily  less 
than  20  p.  m.  protein,  although  in  certain  cases  the  quantity  of  protein 
may  rise  to  35  p.  m.  In  carcinomatous  peritonitis  it  may  have  a  cloudy, 
dirty-gray  appearance,  due  to  its  richness  in  form-elements  of  various  kinds. 
The  specific  gra\'ity  is  then  higher,  the  quantity  of  solids  greater,  and  it 
often  coagulates  spontaneously.  In  inflammatory  processes  it  is  straw-  or 
lemon-yellow  in  color,  somewhat  cloudy  or  reddish,  due  to  leucocytes  and 
red  blood-corpuscles,  and  from  great  richness  in  leucocytes  it  may  appear 
more  like  pus.  It  coagulates  spontaneously  and  may  be  relatively  richer  in 
solids.  It  contains  regularly  30  p.  m.  or  more  protein  (although  exceptions 
with  less  protein  occur),  and  may  have  a  specific  gravity  of  1.030  or  above. 
On  account  of  the  rupture  of  a  chylous  vessel,  the  dropsical  fluid  may  be  rich 
in  very  finely  emulsified  fat  (chylous  ascites).  In  such  cases  3.86-10.30 
p.  m.  fat  has  been  found  in  the  dropsical  fluid  (Guinochet,  Hay  i),  and  even 
17-43  p.  m.  has  been  found  by  Minkowski. 

As  first  shown  by  Gross,  an  ascitic  fluid  may  have  a  chylous  appearance 
without  the  presence  of  fat,  i.e.,  pseudochylous.  The  cause  of  the  chylous 
properties  of  a  transudate  is  not  known,  although  numerous  investigators, 
such  as  Gross,  Bernert,  Mosse,  and  Strauss,  have  studied  the  subject; 
several  observations,  however,  seem  to  show  that  it  is  connected  with  the 
amount  of  lecithin  contained  therein.  In  a  case  investigated  by  H.  Wolff  2 
the  oleic-acid  ester  of  cholesterin  was  combined  either  chemically  or  molecu- 
larly  with  the  euglobulin. 

By  admixture  of  ascitic  fluid  with  the  fluid  from  an  ovarian  cyst  the 
former  may  sometimes  contain  pseudoraucin  (see  Chapter  XIII).  There 
are  also  cases  in  which  the  ascitic  fluid  contains  mucoids  which  may  be 
precipitated  by  alcohol  after  removal  of  the  proteins  by  coagulation  at 
boiling  temperature.  Such  mucoids,  which  yield  a  reducing  substance  on 
boiling  with  acids,  have  been  found  by  Hammarsten  in  tuberculous  peri- 
tonitis and  in  cirrhosis  hepatis  syphilitica  in  men.  According  to  the 
investigations  of  Paijkull,  these  substances  seem  to  occur  often  and  perhaps 
habitually  in  the  ascitic  fluids. 


*  Guinochet,  see  Strauss,  Arch,  de  Physiol.,  18.     See  Maly's  Jahresber.,  16,  475. 

^  Gross,  Arch.  f.  exp.  Path.  u.  Pharm.,  44;  Bernert,  ibul.,  49;  Mosse,  Leyden's 
Festschrift,  1901;  Strauss,  cited  in  Biochem.  Centralbl.,  1,  4.37;  Wolff,  Hofmeister's 
Beitrjise,  5. 


HYDROCELE  AND  SPERMATOCELE  FLUIDS.  263 

As  the  quantity  of  protein  in  ascitic  fluids  is  dependent  upon  the  same 
factors  as  in  other  transudates  and  exudates,  it  is  sufficient  to  give  tlie 
following  example  of  the  composition,  taken  from  Berxheim's  ^  treatise. 
The  results  are  expressed  in  1000  parts  of  the  fluid: 

Max.  Min.  Mean. 

Cirrhosis  of  the  liver 34.5         5.6  9.69—21.06 

Bright's  disease 16.11  10.10  5.6—10.36 

Tuberculous  and  idiopathic  peritonitis.  .  .   55.8  18.72  30.7  — 37.95 

Carcinomatous  peritonitis 54.20  27.00  35. 1   — 58.96 

Joachim  found  the  highest  relative  globulin  amounts  and  lowest  albumin 
percentages  in  cirrhosis;  in  carcinoma,  on  the  contrary,  the  lowest  globulin  and 
the  highest  albumin.  The  values  in  cardiac  stagnation  stand  between  the  cir- 
rhosis and  carcinoma  percentages. 

Urea  has  also  been  found  in  ascitic  fluids,  sometimes  only  as  traces,  some- 
times in  larger  quantities  (4  p.  m.  in  albuminuria),  also  uric  acid,  allantoin  in 
cirrhosis  of  the  liver  (Mosc-vtelli),  xanthine,  creatine,  cholesterin,  sugar,  diastatic 
and  proteolytic  enzymes,  and  according  to  Hamburger  '  also  a  lipase. 

Hydrocele  and  Spermatocele  Fluids.  These  fluids  differ  essentially 
from  each  other  in  various  ways.  The  hydrocele  fluids  are  generally  colored 
light  or  dark  yellow,  sometimes  brownish  with  a  shade  of  green.  They 
have  a  relatively  higher  specific  gTa\ity,  1.016-1.026,  with  a  variable  but 
generally  higher  amount  of  solids,  an  average  of  60  p.  m.  They  sometimes 
coagulate  spontaneously,  sometimes  only  after  the  addition  of  fibrin  ferment 
or  blood.  They  contain  leucocytes  as  chief  form-elements.  Sometimes 
they  contain  smaller  or  larger  amounts  of  cholesterin  crystals. 

The  spermatocele  fluids,  on  the  contrary,  are  as  a  rule  colorless,  thin,  and 
cloudy  like  water  mixed  with  milk.  They  sometimes  have  an  acid  reaction. 
They  have  a  lower  specific  gravity,  1.006-1.010,  a  lower  amount  of  solids — 
an  average  of  about  13  p.  m. — and  do  not  coagulate  either  spontaneously 
or  after  the  addition  of  blood.  They  are,  as  a  rule,  poor  in  protein  and 
contain  spermatozoa,  cell-detritus,  and  fat-globules  as  form-constituents.  To 
show  the  unequal  composition  of  these  fcwo  kinds  of  fluids  we  will  give  the 
average  results  (calculated  in  parts  per  1000  parts  of  the  fluid)  of  seven- 
teen analyses  of  hydrocele  fluids  and  four  of  spermatocele  fluids  made  bjy 
Hammarstex.3 

Hydrocele.  Spermatocele. 

Water 938 .  85  986 .  83 

Sohds 61.15  12.17 

Fibrin 0.59  

Globulin 13.25  0.59 

Seralbumin 35.94  1.82 

Ether  extractive  bodies 4 .  02  "I 

Sohible  salts 8.60}.  10.76 

Insohible  salts 0 .  66  J 

'  1.  c.     As  it  was  impossible  to  derive  mean  figures  from  those  given  by  Bernheim, 
the  author  has  given  the  maximum  and  minimum  of  the  averages  given  by  him. 
2  Arch.  f.  (Anat.  u.)  Physiol.,  1900,  433. 
'  Upsala  Lakaref.  Forh.,  14,  and  Maly's  Jahresber.,  8,  347. 


264  CHYLE,  LY^IPH,  TRANSUDATES  AND  EXUDATES. 

In  the  hj'drocele  fluid  traces  of  urea  and  a  reducing  substance  have  beau 
found,  and  in  a  few  cases  also  succinic  acid  and  inosite.  A  hydrocele  fluid  may, 
according  to  Devillard,'  sometimes  contain  paralbumin  or  metalbumin  (?). 
Cases  of  chylous  hydrocele  are  also  knowTi. 

Cerebrospinal  Fluid.  The  cerebrospinal  fluid  is  thin,  water-clear,  of 
low  specific  gravity,  1. 007-1. OOS.  The  spina  bifida  fluid  is  very  poor  in 
solids,  8-10  p.  m.,  with  only  0.19-1.6  p.  m.  protein.  The  fluid  of  chronic 
hydrocephalus  is  somewhat  richer  in  solids  (13-19  p.  m.)  and  proteins. 
According  to  Halliburton  the  protein  of  the  cerebrospinal  fluid  is  a 
mixture  of  globulin  and  proteose;  occasionally  some  peptone  occurs,  and 
more  rarely,  in  special  cases,  seralbumin  appears.  The  statements  of 
Halliburton  on  the  occurrence  of  ^proteose  do  not  coincide  with  the  ob- 
servations of  other  investigators  (Panzer,  Salkowski^).  In  general 
paralysis  Halliburton  and  Mott  have  obtained  a  nucleoproteid  in  the 
cerebrospinal  fluid.  Choline  occurs  in  several  diseases,  as  in  general  paral- 
ysis, brain-tumors,  tabes  dorsalis,  and  epilepsy  (Halliburton  and  Mott, 
DoNATH^).  Dextrose,  or  at  least  a  fermentable  sugar,  occurs  habitually 
in  the  cerebrospinal  fluid,  while  the  statements  of  Halliburton  as  to  the 
occurrence  of  a  substance  similar  to  pyrocatechin  could  not  be  substantiated 
by  Nawratzki,^  and  hence  this  substance  does  not  exist  in  all  cerebro- 
spinal fluids.  Urea  occurs  in  cerebrospinal  fluids,  but  not  alwaj's.  The 
variable  relationship  between  potassium  and  sodium  ^  is  probably  due, 
according  to  Salkowski,  to  the  absence  or  presence  of  fever  during  the 
formation  of  the  exudate;  the  amount  of  potassium  is  high  in  the  acute 
cases  and  low  in  the  chronic  ones.  According  to  Cavazzani,^  who  has  es- 
pecially studied  the  cerebrospinal  fluids,  the  alkalinity  of  these  fluids  is 
considerably  less  than  that  of  the  blood  and  independent  of  this  last  fluid. 
For  this  and  several  other  reasons  Cavazzani  draws  the  conclusion  that  the 
cerebrospinal  fluid  is  formed  by  a  true  secretory  process. 

Aqueous  Humor.  This  fluid  is  clear,  alkaline  towards  litmus,  and  has 
a  specific  gravity  of  1.003-1.009.  The  amount  of  solids  is  on  an  average 
13  p.  m..  and  the  amount  of  proteins  only  0.8-1.02  p.  m.  The  protein  con- 
sists of  seralbumin  and  globulin  and  very  little  fibrinogen.  According  to 
Gruenhagen  it  contains  paralactic  acid,  another  dextrogyrate  substance, 

1  Bull.  Soc.  chim.,  49,  617. 

-  Halliburton's  Text-book;  Panzer,  Wien.  klin.  Wochenschr.,  1899;  Salkowski, 
Jaffe  Festschrift,  26.5. 

^  Halliburton  and  Mott,  Phil.  Transact.  Roy.  Soc.  London,  Series  B,  191;  Donath, 
Zeitschr.  f.  physiol.  Cbem..  39  and  42;   see  also  Mansfield,  ibid.,  42. 

*  Zeitschr.  f.  physiol.  Chem.,  23.     See  also  Rossi,  ibid..  39  (literature). 

*  See  Salkowski,  1.  c.  New  quantitative  analyses  of  cerebrospinal  and  hydro- 
cephalus fluids  may  be  found  in  the  cited  works  of  Xawratzki,  Panzer,  and  Salkowski. 

'See  Maly's  Jahresber.,  22,  346,  and  Centralbl.  f.  Physiol.,  15,  216. 


SYNOVIAL   FLUID.  265 

and  a  reditcing  body  which  is  not  similar  to  dextrose  or  dextrin.  Pautz  ^ 
found  urea  and  sugar  in  the  aqueous  humor  of  oxen. 

Blister-fluid.  The  content  of  blisters  caused  by  burns,  and  of  vesicatory 
blisters  and  the  blisters  of  the  pemphigus  chronicus,  is  generally  a  fluid 
rich  in  solids  and  proteins  (40-65  p.  m.).  This  is  especially  true  of  the 
contents  of  vesicators^  blisters.  In  a  burn-blister  K.  ^Iorxer^  found  50.31 
p.  m.  proteins,  among  which  were  13.59  p.  m.  globulin  and  0.11  p.  m.  fibrin. 
The  fluid  contains  a  substance  which  reduces  copper  oxide,  but  no  pyro- 
catechin.  The  fluid  of  the  pemphigus  is  alkaline  in  reaction.  A  wound 
secretion  collected  by  Liebleix  ^  under  aseptic  conditions  was  alkaline  in 
reaction  and  contained  less  protein  than  the  blood-serum.  It  formed 
a  slight  fibrin  clot  and  contained  proteoses  only  at  first  or  at  the  beginning 
of  the  abscess  formation.  As  the  wound  liealed,  the  relationship  between 
the  globulin  and  albumin  changed,  and  on  the  third  day  of  the  healing 
the  quantity  of  albumin  was  at  least  nine  tenths  of  the  total  protein. 

The  fluid  of  subcutaneous  oedema.  This  is,  as  a  rule,  xery  poor  in 
solids,  purely  serous,  does  not  contain  fibrinogen,  and  has  a  specific  gra^■ity 
of  1.005-1.013.  The  quantity  of  proteins  is  in  most  cases  lower  than  10 
p.  m., — according  to  Hoffil^xx  1-8  p.  m., — and  in  serious  affections  of 
the  kidneys,  generally  with  amyloid  degeneration,  less  than  1  p.  m.  has  been 
shown  (HoFFiL\NN  *) .  The  oedematous  fluid  also  habitually  contains  urea, 
1-2  p.  m.,  and  sugar. 

The  FLUID  OF  THE  ECHixococcus  cvst  Is  related  to  the  transudates  and  is  poor 
in  proteins.  It  is  thin  and  colorless,  and  has  a  specific  gravity  of  1.005-1.015. 
The  quantity  of  solids  is  14-20  p.  m.  The  chemical  constituents  are  sugar  (2.5 
p.  m.),  inosite,  traces  of  urea,  creatin-e,  succinic  acid,  and  salts  (8.3-9.7  p.m.). 
Proteins  are  found  only  in  traces,  and  then  only  after  an  inflammatory  irritation. 
In  the  last-mentioned  case  7  p.  m.  proteins  have  been  found  in  the  fluid. 

The  Synovial  Fluid  and  Fluid  in  Synovial  Cavities  around  Joints,  etc. 

The  synovia  is  hardly  a  transudate,  but  it  is  often  discussed  in  an  appendix 
to  the  transudates. 

The  syno\-ia  is  an  alkaline,  sticky,  fibrous,  yellowish  fluid  which  is 
cloudy,  from  the  presence  of  cell-nuclei  and  the  remains  of  destroyed  cells,  but 
is  also  sometimes  clear.  It  contains  also,  besides  proteins  and  salts,  a  mucin 
substance,  synoviamucin  (v.  Holst^).  In  pathological  synovia  Haa^l^r- 
STEN  has  found  a  mucin-like  substance  which  is  not  mucin.  It  behaves 
like  a  nucleoalbumin  or  a  nucleoproteid  and  gives  no  reducing  substance 
on  boiling  with  acids.     Salkowski  ^  also  found  a  mucin-like  substance  in  a 

'  Gruenhagen,  Pfliiger's  Arch.,  43;    Pautz,  Zeitschr.  f.  Biologie,  31. 

^  Skand.  Arch.  f.  Pliysiol.,  5. 

^  Habilitationsschrift  Prag,  1902,  printed  by  H.  Laupp,  Tiibingen. 

*  Deutsch.  Arch.  f.  klin.  Med.,  44. 
'  Zeitschr.  f.  phj-siol.  Chem.,  43. 

•  Hammarsten,  Maly's  Jahresber.,  12;   Salkowski.  Virchow's  Arch.,  131. 


266  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

pathological  synovial  fluid,  which  was  neither  mucin  nor  nucleoalbumin. 
He  called  the  substance  synovin. 

The  composition  of  synovia  is  not  constant,  but  is  different  in  rest  and 
in  motion.  In  the  last-mentioned  case  the  quantity  of  fluid  is  less,  but  the 
amount  of  the  mucin-like  body,  of  proteins,  and  of  the  extractive  bodies  is 
greater,  while  the  quantity  of  salts  is  diminished.  This  may  be  seen  from 
the  following  analyses  by  Frerichs.^     The  figures  represent  parts  per  1000. 

I.  Synovia  from      II.  Synovia  from 
a  ytall-fed  ox.  a  Field-fed  ox. 

Water 969.9  948.;') 

Solids 30.1  51.5 

Mucin-like  body 2.4  5.6 

Albumin  and  extractives 15 . 7  35. 1 

Fat 0.6  0.7 

Salts 11.3  9.9 

The  synovia  of  new-born  babes  corresponds  to  that  of  resting  animals. 
The  fluid  of  the  bursse  mucosas,  as  also  the  fluid  in  the  synovial  cavities 
around  joints,  etc.,  is  similar  to  synovia  from  a  qualitative  standpoint. 

m.   Pu.s. 

Pus  is  a  yellowish-gray  or  yellowish-green,  creamy  mass  of  a  faint  odor 
and  an  unsavory,  sweetish  taste.  It  consists  of  a  fluid,  the  pus-serum,  in 
which  solid  particles,  the  pus-cells,  swim.  The  number  of  these  cells  varies 
so  considerably  that  the  pus  may  at  one  time  be  thin  and  at  another  time 
so  thick  that  it  scarcely  contains  a  drop  of  serum.  The  specific  gravity, 
therefore,  may  also  greatly  vary,  namely,  between  L020  and  1.040,  but 
ordinarily  it  is  1.031-1.033.  The  reaction  of  fresh  pus  is  generally  alkaline, 
but  it  may  become  neutral  or  acid  from  a  decomposition  in  which  fatty 
acids,  glycerophosphoric  acid,  and  also  lactic  acid  are  formed.  It  may 
become  strongly  alkaline  when  putrefaction  occurs  with  the  formation  of 
ammonia. 

In  the  chemical  investigation  of  pus,  the  pus-serum  and  the  pus-corpus- 
cles must  be  studied  separately. 

Pus-serum.  Pus  does  not  coagulate  spontaneously  nor  after  the  addi- 
tion of  defibrinated  blood.  The  fluid  in  which  the  pus-corpuscles  are 
suspended  is  not  to  be  compared  with  the  blood-plasma,  but  rather  with 
the  serum.  The  pus-serum  is  pale  yellow,  yellowish  green,  or  brownish 
yellow,  and  has  an  alkaline  reaction  towards  litmus.  It  contains,  for  the 
most  part,  the  same  constituents  as  the  blood-serum;  but  sometimes  be- 
sides these — when,  for  instance,  the  pus  has  remained  in  the  body  for  a 
long  time — it  contains  a  nucleoalbumin  or  a  nucleoproteid  which  is  precipi- 
tated by  acetic  acid  and  is  soluble  with  great  difficulty  in  an  excess  of  the 
acid  (pi/in  of  the  older  authors).  This  nucleoalbumin  seems  to  be  formed 
'  Wagner's  Handworterbuch,  3,  Aht.,  41  ri3. 


PUS.  267 

from  the  hyaline  substance  of  the  pus-cells  by  maceration.  The  pus-serum 
contains,  moreover,  at  least  in  many  cases,  no  fibrin  ferment.  According 
to  the  analyses  of  Hoppe-Seyler  ^  the  pus-serum  contains  in  1000  parts : 

I.  II. 

Water 913 .  70  905 .  65 

Solids 80 .  30  94 .  35 

Proteins 63.23  77.21 

Lecithin 1 .50  0.56 

Fat 0.26  0.29 

Cholesterin 0.53  0.87 

Alcohol  extractives 1  .  52  0 .  73 

Water  extractives 1 1 .  53  6 .  92 

Inorganic  salts 7 .  73  7 .  77 

The  ash  of  pus-serum  has  the  following  conaposition,  calculated  to  100& 
parts  of  the  serum : 

I.  II. 

NaCl 5.22  5.39 

Na„S04 0.40  0.31 

Na,HPO, 0.98  0.46 

NalCOg 0.49  1.13 

CagCPOJa 0.49  0.31 

MgjCPO,)., 0.19  0.12 

PC),  (in  eicess) 0.05 

The  pus-corpuscles  are  generally  thought  to  consist  in  great  part  of 
emigrated  white  blood-corpuscles,  and  their  chemical  properties  have 
therefore  been  given  in  discussing  these.  The  molecular  granules,  fat-glob- 
ules, and  red  blood-corpuscles  are  considered  rather  as  casual  form-elements. 

The  pus-cells  may  be  separated  from  the  serum  by  centrifugal  force,  ot 
by  decantation  directly  or  after  dilution  with  a  solution  of  sodium  sulphate 
in  water  (1  vol.  saturated  sodium-sulphate  solution  and  9  vols,  water),  and 
then  washed  by  this  same  solution  in  the  same  manner  as  the  blood-cor- 
puscles. 

The  chief  constituents  of  the  pus-corpuscles  are  proteins  of  wUch 
the  largest  portion  seems  to  be  a  nucleoproteid  which  is  insoluble  in 
water  and  which  expands  into  a  tough,  slimy  mass  when  treated  with  a  10 
per  cent  common-salt  solution.  This  protein  substance,  which  is  soluble  in 
alkali  but  is  quickl}^  changed  thereby,  is  called  Rovida's  hyaline  substance, 
and  the  property  of  the  pus  of  being  converted  into  a  slime-like  mass  by  a 
solution  of  common  salt  depends  on  this  substance.  Besides  this  substance 
the  pus-cells  contain  also  a  globulin  which  coagulates  at  48-49°  C,  as  well 
as  serglobulin  (?),  seralbumin,  a  substance  similar  to  coagulated  protein 
(Miescher),  and  lastly  peptone  or  proteose  (Hofmeister^).  It  is  very 
remarkable  that  no  nucleohistone  or  histone  has  been  detected  in  the  pus- 
cells. 

'  Med.-cliem.  Untersuch.,  490. 

2  Miescher  in  Hoppe-Seyler's  Med.-chem.  Untersuch.,  441;  Hofmeister,  Zeitschr.  f. 
physiol.  Chem.,  4. 


268     CHYLE,  LY:\IPH,  TRANSUDATES  AND  EXUDATES. 

There  are  also  found  in  the  protoplasm  of  the  pus-cells,  besides  the  pro- 
teins, lecithin,  cholesterin,  xanthine  bodies,  fat,  and  soaps.  Hoppe-Seyler 
has  found  cerehrin,  a  decomposition  product  of  a  protagon-like  substance, 
in  pus  (see  Chapter  XII).  Kossel  and  Freytag  ^  have  isolated  from  pus 
two  substances,  pyosin  and  pyogenin,  which  belong  to  the  cerebrin  group 
(see  Chapter  XII).  Hoppe-Seyler  ^  claims  that  glycogen  appears  only  in 
the  li\'ing,  contractile  white  blood-cells  and  not  in  the  dead  pus-corpuscles. 
Several  other  investigators  have  nevertheless  found  glycogen  in  pus.  The 
cell-nucleus  contains  nuclein  and  nucleoproteids.  ^Iandel  and  Levene  ^ 
have  shuwn  the  occurrence  of  glucothionic  acid  in  the  pus-cells. 

In  regard  to  the  occurrence  of  enzymes  in  the  pus-cells  it  must  be  re- 
marked that  neither  thrombin  nor  prothrombin  is  found  therein,  although 
these  bodies  are  generally  considered  as  being  derived  from  the  leucocytes 
and  can  also  be  obtained  from  the  thymus  leucocytes.  The  occurrence 
in  the  pus-cells,  besides  catalases  and  oxidases,  of  a  proteolytic  enzyme  is 
of  great  interest.  It  is  not  only  important  for  the  intracellular  digestion 
and  for  the  amount  of  proteoses  in  the  pus-cell,  but  also  for  the  solution  of 
the  fibrin  clot  and  pneumonic  infiltrations  (Fr.  ]Muller,  O.  Simon  *). 

The  mineral  constituents  of  the  pus-corpuscles  are  potassium,  sodium, 
calcium,  magnesium,  and  iron.  A  part  of  the  alkalies  exists  as  chlorides, 
and  the  remainder,  as  well  as  the  chief  part  of  the  other  bases,  exists  as 
phosphates. 

The  quantitative  composition  of  the  pus-cells  from  the  anal5'ses  of 
Hoppe-Seyler  is  as  follows,  in  parts  per  1000  of  the  dried  substance: 

I.  II. 

Proteins 137 .62  1 

Nuclein 342.57  [685.85       673.69 

Insoluble  bodies 205 .  66  J 

Lecithin 1  i  <  o  oo  75 .  64 

Fat ;  ^-^'^-^^  75.00 

Cholesterin 74.00  72.83 

Cerebrin 51 .99  \  mo  84. 

Extractive  bodies 44.33/  lu^.a* 

MINERAL    SUBSTANCES   IN    1000    PARTS    OF    THE    DRIED    SUB.STANCE. 

NaCl 4.34 

CagCPOJ, 2.05 

Mg3(P0j; 1.13 

FePO^ 1 .06 

PO, 9.16 

Na 0.68 

K Traces    (?) 

'  Zeitschr.  f.  physiol.  Chem.,  17,  452. 
^  Hoppe-Seyler,  Physiol.  Chem.,  790. 
'  Biochem.  Zeitschrift.  4. 

■•  Fr.  Miiller,  Verhandl.  Nat.  Gesellsch.  zu  Basel,  1901;  O.  Simon,  Deutsch.  Arch, 
f.  klin.  Med.,  70. 


LY:\rPHATIC   GLAXDS.  269 

MiEscHER  has  obtained  other  results  for  the  alkali  compounds,  namely, 
potassium  phosphate  12,  sodium  phosphate  6.1,  earthy  phosphate  and  iron 
phosphate  4.2,  sodium  chloride  1.4,  and  phosphoric  acid  combined  with  organic 
substances  3.14-2.03  p.  m. 

In  pus  from  congested  abscesses  which  have  stagnated  for  some  time 
occur  peptone  (proteose),  leucine  and  tyrosine,  free  fatty  acids  and  volatile 
fatty  acids,  such  as  formic  acid,  butyric  acid  and  valerianic  acid.  There  are 
also  found  chondrin  (?)  and  glutin  (?),  wea,  dextrose  (in  diabetes),  hile- 
pigments  and  bile-acids  ((in  catarrhal  icterus). 

As  more  specific  but  not  constant  constituents  of  the  pus  must  be  men- 
tioned the  following:  pyin,  which  seems  to  be  a  nucleoproteid  precipitable 
by  acetic  acid,  and  also  pyinic  acid  and  chlorrhodinic  acid,  which  have  been 
so  little  studied  that  they  cannot  be  more  fully  treated  here. 

In  many  cases  a  blue,  more  rarely  a  green,  color  has  been  observed  in 
the  pvis.  This  depends  on  the  presence  of  micro-organisms  {Bacillus  pyo- 
cyaneiis).  From  such  pus  Fordos  and  Lucre  ^  have  isolated  a  crystalline 
blue- pigment,  pyocyanin,  and  a  yellow  pigment,  pyoxanthose,  which  is  pro- 
duced from  the  first  by  oxidation. 


Appendix. 

LYMPHATIC  GLANDS,  SPLEEN,  ETC. 

The  Lymphatic  Glands.  The  cells  of  the  lymphatic  glands  are  found 
to  contain  the  protein  substances  occurring  generally  in  cells  (Chapter  V, 
pages  141  and  142).  According  to  Baxg^  they  also  contain  histone  nucleates 
(jiucleohi stone) .  but  in  smaller  amounts  and  of  a  different  variety  from  ihe 
better-studied  nucleohistone  from  the  thymus  gland.  Proteoses  occur  as 
products  of  autolysis.  By  a  lengthy  autotysis  of  lymph-glands  Reh  ^  found 
ammonia,  tyrosine,  leucine  (somewhat  scanty),  thymine,  and  uracil  among 
the  cleavage  products.  Besides  the  other  ordinary  tissue  constituents,  such 
as  collagen,  reticulin,  elastin,  and  nuclein,  there  occur  in  the  lymphatic 
glands  also  cholesterin,  fat,  glycogen,  sarcolactic  acid,  xanthine  bodies,  and 
leucine.  In  the  inguinal  glands  of  an  old  woman  Oidt^l\xn  found  714.32 
p.  m.  water,  2S4.5  p.  m.  organic  and  1.16  p.  m.  inorganic  substances.  In 
the  cells  of  the  mesenteral  lymphatic  glands  of  oxen  Bang^  found  804.1 
p.  m.  water,  195.9  p.  m.  solids,  137.9  total  proteins.  6.9  p.  m.  histone  nucleate 
10.6  p.  m.  nucleoproteid,  47.6  p.  m.  bodies  soluble  in  alcohol,  and  10.5  p.  m. 
mineral  constituents. 

'  Fordo.«;,  Corapt.  rend.,  ol  and  56;    Litcke,  Arch.  f.  kiln.  Chirurg.,  3;    Boland,  Cen- 
tralbl.  f.  Bakt.  u.  Parasit.,  I,  25. 

'  Studier  over  Nucleoproteider,  Kristiania,  1902,  and  Hofmeister's  Beitrage,  i. 
^  Hofmeister's  Beitrage,  3. 
M.  c. 


270  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

The  Thymus.  The  cells  of  this  gland  are  veiy  rich  in  nuclein  bodies 
and  relatively  poor  in  the  ordinan,'  proteins,  but  their  nature  has  not  been 
closely  studied.  The  chief  interest  is  attached  to  the  nuclein  substances. 
KossEL  and  Liliexfeld  first  prepared  from  the  waterj-  extract  of  the 
gland,  by  precipitating  ^dth  acetic  acid  and  then  further  purifying,  a  protein 
substance  which  has  been  generally  called  niideohistone.  By  the  action 
of  dilute  hydrochloric  acid  upon  nucleoliistone  it  splits,  according  to  these 
investigators,  into  histone  and  leuconuclein.  The  leuconuclein  is  a  true 
nuclein;  hence  it  is  a  nucleic-acid  compound  with  protein  which  is  relatively 
poor  in  protein  and  rich  in  phosphorus.  The  more  recent  investigations 
of  Bang,  ^La.lexgreau.  and  Huisk.\mp  ^  upon  nucleohistone  all  show 
that  tliis  nucleoproteid  is  not  a  unit  substance  but  a  mixture  of  at  least 
two  bodies.  The  views  of  the  investigators  mentioned  differ  quite  esseu' 
tially  from  one  another  as  to  the  nature  of  these  bodies,  but  this  is  partly 
due  to  the  different  methods  used  by  them  and  partly  to  the  ready  change- 
ability of  the  substances  in  question. 

Besides  the  real  nucleohistone,  B-nucleoalbuminof  Malengreau,  Liliex- 
feld's  histone  contains  a  second  nucleoproteid  which  Baxg  and  Huiskamp 
call  simple  nucleoproteid,  while  Malexgreau  designates  it  A-nucleoalbumin. 
This  protein,  which  contains  only  about  1  per  cent  phosphorus  and  which 
is  possibly  identical  with  the  nucleoproteid  found  by  Liliexfeld  in  the 
thymus,  yields  a  nuclein,  but  no  free  nucleic  acid,  on  cleavage.  As  second 
cleavage  product  it  yields,  according  to  Malengreau,  the  A-histone,  which 
can  be  readily  precipitated  by  magnesium  and  ammonium  sulphates  from 
the  ordinary  B-liistone  of  the  thymus  gland.  The  occurrence  of  A-histone 
in  the  gland  has  been  verified  by  Bang,  and  according  to  Bang  and  Huis- 
K-\MP  the  A-liistone  is  not  derived  from  the  nucleoproteid,  as  these  inves- 
tigators claim  that  it  yields  no  histone.  According  to  Baxg  the  nucleo- 
proteid yields  only  an  albuminate,  besides  the  nuclein,  as  cleavage  products. 

The  true  nucleoliistone,  which  is  much  richer  in  phosphorus  (the  calcium 
salt  containing,  according  to  Baxg,  on  an  average  5.23  per  cent  P),  yields 
ordinary'  histone  as  one  cleavage  product  and  free  nucleic  acid  as  the  other, 
according  to  the  unanimous  opinion  of  the  above-mentioned  investigators. 
According  to  Baxg,  who.se  statements  on  this  point  have  been  substantiated 
by  ^La.lengreau,  it  splits  on  saturating  with  XaCl  into  nucleic  acid  and 
histone  without  yielding  any  other  protein.  On  this  account  B.an'G  does  not 
consider  this  body  as  nucleohistone  in  the  ordinary-  sense,  i.e.,  not  as  a  nucleo- 
proteid, but  as  a  histone  nucleate.  The  nucleohistone  behaves  like  an  acid, 
who.se  salts,  especially  the  calcium  salt,  have  been  closely  studied  by  Huis- 

'  Lilienfeld,  Zeitschr.  f.  physiol.  Chem.,  IN;  Ko.ssel,  ibid.,  30  and  31;  Bang,  ibid., 
30  and  31.  See  also  Arch.  f.  Math,  og  Naturvidenskab,  25,  Kristiania,  1902,  and 
Hofmeister's  Reitrage,  1  and  4;  Malengreau,  La  Cellule,  17  and  19;  Huiskamp,  Zeit- 
schr. f.  phj'siol.  Chem.,  32,  34,  and  39. 


THYMUS.  271 

KAMP.  On  the  electrolysis  of  a  solution  of  alkali  nucleohistone  in  water  Huis- 
K.\MP  found  also  that  the  nucleohistone  collected  in  traces  at  the  anode,  and 
that  the  sodium  compound  is  therefore  ionized  in  the  solution.  The 
nucleic  acid-calcium-histone  compound  has  been  prepared,,  it  seems,  in  a 
pure  state  by  Bang,  and  he  foimd  the  follo^^■ing  average  composition: 
C  43.69;  Ho.60;  X  16.87;  S0.47;  P5.23;  Ca  1.71  per  cent.  The  question 
as  to  what  compound  contains  the  A-histone  remains  to  be  investigated. 

The  nucleohistone  prepared  by  Huiskamp's  method  by  precipitating  withCaCli 
is,  according  to  him,  a  mbcture  of  two  nucleohistones,  of  which  one,  the  a-nucleo- 
histone,  contains  4.5  per  cent  phosphorus,  and  the  other,  .-J-nucleohistone,  contains, 
on  the  contrary,  only  in  round  numbers  3  per  cent  phosphorus.^  As  the  two 
nucleohistones  are  poorer  in  phosphorus  than  the  nucleic  acid-histone  compound 
analyzed  by  Bang,  and  as  Huiskamp  on  cleavage  of  his  preparation  did  not,  like 
Bang  and  Malexgreau,  obtain  pure  nucleic  acid,  it  is  still  a  question  whether 
Huiskamp  was  working  with  sufficiently  pure  substances. 

In  regard  to  the  methods  used  by  the  above  investigators  in  the  isola- 
tion of  the  bodies  in  question  we  must  refer  to  the  oridnal  publications. 

In  connection  with  the  so-called  nucleohistone,  attention  must  be  called  to 
tissue  fibrinogen  and  cdl  fibrinogen,  which  are  compound  proteins,  and  are  claimed 
by  certain  mvestigators  to  stand  in  close  relation  to  the  coagulation  of  the  blood. 
These  may  be  in  part  nucleoproteids  and  in  part  also  nucleohistones.  To  this 
same  group  belong  also  the  important  cell  constituents  described  by  Alex. 
Schmidt-  and  called  cytoglobin  and  preglobulin.  The  cytoglobin,  which  is 
soluble  in  water,  may  be  considered  as  the  alkali  compound  of  preglobulin.  The 
residue  of  the  cells  left  after  complete  extraction  with  alcohol,  water,  and  salt 
solution  has  been  called  cytin  by  Alex.  Schmidt. 

Besides  the  above-mentioned  and  the  ordinary'  bodies  belonging  to  the 
connective-tissue  group,  small  quantities  of  jat,  leucine,  succinic  acid,  lactic 
add,  sugar,  and  traces  of  iodothyrin  are  present.  According  to  Gautier^ 
arsenic  also  occtirs  in  ver\-  small  amounts,  and  no  doubt  here  as  well  as  in 
other  organs  it  is  related  to  the  nuclein  substances.  The  richness  in  nuclein 
bodies  explaias  the  occurrence  of  large  quantities  of  purine  bases,  chiefly 
adenine,  whose  quantity,  according  to  Kossel  and  Schixdler,-*  is  1.79  p.  m. 
in  the  fresh  organ  and  19.19  p.  m.  in  the  dr\'  substance.  The  bodies  thymine 
and  uracil  (?)  obtained,  besides  lysine  and  ammonia,  by  Kutscher,  as  prod- 
ucts of  autodigestion  of  the  gland,  probably  have  a  similar  origin.  Liliex- 
FELD  ^  has  found  inosite  and  protagon  in  the  cells  of  the  thymus.  Among 
the  enzymes,  besides  arginase,  guanase.  and  adenase,  we  must  especially 
mention  the  enzyme  studied  by  Joxes.  wliich  acts  like  a  nuclease,  splitting 
off  phosphoric  acid  and  ])urine  bases  from  the  nucleoproteids.     This  enzyme, 

»  Zeitschr.  f.  physiol.  Chem.,  39. 

^  See  foot-note  5,  p.  141. 

3  Compt.  rend.,  129. 

*  Zeitschr.  f.  physiol.  Chem.,  13. 

-Kutscher,  ibid.,  34;    Lilienfeld,  ibid.,  18. 


272  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

contrary  to  trypsin,  acts  best  in  acid  liquids  and  is  readily  destroyed  by 
alkalies  at  body  temperature.^  The  quantitative  composition  of  the 
lymphocytes  of  the  th3^mus  of  a  calf  is,  according  to  Lilienfeld's  analysis, 
as  follows.     The  results  are  given  in  1000  parts  of  the  dried  substance. 

Proteids 17.7 

Leuconuclein 687 . 9 

Histone S6 . 7 

Lecithin 75.1 

Fat 40 . 2 

Cholesteiin 44 . 0 

Glycogen S .  0 

The  dried  substance  of  the  leucocytes  amounted  to  an  average  of  114.9 
p.  m.  Potassium  and  phosphoric  acid  are  prominent  mineral  constituents. 
LiLiENFELD  fouud  KH0PO4  ainongst  the  bodies  soluble  in  alcohol. 

Attention  must  be  called  to  the  analyses  of  Bang  2  which  show  that  the 
thymus  contains  about  the  same  quantity  of  nucleoproteid,  but  about  five 
times  as  much  histone  nucleate  as  the  lymphatic  glands — calculated  in  both 
cases  upon  the  same  amount  of  dry  substance.  Oidtmann  ^  found  807.06 
p.  m.  water,  192.74  p.  m.  organic  and  0.2  p.  m.  inorganic  substances  in  the 
gland  of  a  child  two  weeks  old. 

The  Spleen.  The  pulp  of  the  spleen  cannot  be  freed  from  blood.  The 
mass  which  is  separated  from  the  spleen  capsule  and  the  structural  tissue 
Iw  pressure  and  which  ordinarily  serves  as  material  for  chemical  investiga- 
tions is  therefore  a  mixture  of  blood  and  spleen  constituents.  For  this 
reason  the  proteins  of  the  spleen  are  little  known.  The  nucleoproteid 
isolated  by  Levene  and  Mandel  is  to  be  considered  as  a  true  spleen  con- 
stituent. The  ferruginous  albuminate  has  been  considered  as  a  spleen 
constituent  for  a  long  time,  and  especially  also  a  protein  substance  which 
does  not  coagulate  on  boiling,  and  which  is  precipitated  by  acetic  acid 
and  yields  an  ash  containing  much  phosphoric  acid  and  iron  oxide.* 

The  pulp  of  the  spleen,  when  fresh,  has  an  alkaline  reaction,  but  quickly 
turns  acid,  due  partly  to  the  formation  of  free  paralactic  acid  and  partly 
perhaps  to  glycei'ophosphoric  acid.  Besides  these  two  acids  there  have 
been  found  in  the  spleen  also  volatile  fatty  acids,  as  formic,  acetic,  and 
butyric  acids,  as  well  as  succinic  acid,  neutral  fats,  cholesterin,  traces  of 
leucine,  inosite  (in  ox-spleen),  scyllite,  a  body  related  to  inosite  (in  the  spleen 
of  Plagiostoma),  glycogen  (in  dog-spleen),  uric  acid,  xanthine  bodies,  and 
jecorin.  Levene  has  found  in  the  spleen  a  glucothionic  acid,  i.e.,  an  acid 
which  is  related  to  chondroitin-sulphuric  acid  but  not  identical  therewith, 
and  which  gives  a  beautiful  violet  coloration  with  orcin  and  hydrochloric 

*  Zeitschr.  f.  physiol.  Chem.,  41. 
'1.  c,  Arch.  f.  Math.,  etc. 

'  Cited  from  v.  Gorup-Besanez,  Lehrb.  d.  physiol.  Chem.,  4.  Aufl.,  p.  732. 

*  See  V.  Gorup-Besanez,  Lehrbuch,  4.  Aufl.,  p.  717. 


SPLEEN.  273 

acid.  The  question  whether  this  glucothionic  acid  originates  from  the 
above-mentioned  nucleoproteid  or  from  the  mucoid  substance  has  not  been 
decided  (Levene  and  Mandel^). 

]\Iany  enzymes  are  found  in  the  spleen,  and  certain  of  these  are  of  special 
interest.  To  these  belong  the  uric-acid-forming  enzyme,  the  xanthine  oxidase 
(Burian),  which  occurs  in  the  spleen  of  oxen  and  horses,  but  not  in  man, 
dogs,  and  pigs  (Schittexhelm),  and  which  transforms  the  oxypurines, 
hypoxanthine,  and  xanthine  into  uric  acid;  also  the  hydrolytically  active 
deamidizing  enzymes  guanase  and  adenase  (Levene,  Schittenhelm, 
Jones  and  Partridge,  Jones  and  Winternitz),  by  the  first  of  which 
the  guanine  is  transformed  into  xanthine,  and  the  adenine  into  hypo- 
xanthine by  the  latter.  The  guanase  occurs  also  in  the  spleen  of  the  ox 
and  horse,  but  not  (Jones),  or  only  in  small  amounts  (Schittenhelm),  in 
the  pig-spleen.2  The  spleen  also  contains  two  enzymes,  lienases,  as  shown 
by  Hedin  (and  Rowland),  one  of  which,  the  a-lienase,  acts  chiefly  in 
alkaline  solution,  while  the  other,  >5-lienase,  is  active  only  in  acid  reaction. 
These  enz3^mes  not  only  act  autolytically  upon  the  proteins  of  the  spleen, 
but  they  also  dissolve  fibrin  and  coagulated  blood-serum.  In  the  autolysis 
of  the  spleen  Leathes  found  proteoses,  lysine,  arginine,  histidine,  leucine, 
arainovalerianic  acid,  aspartic  acid,  and  tr}^ptophane  among  the  cleavage 
products.  ScHUMM  ^  found,  in  the  autolysis  of  a  leucsemic  spleen,  besides 
leucine  and  tyrosine  relatively  large  quantities  of  ammonia,  also  r-alanine, 
histidine,  and  lysine  (but  no  arginine),  guanine,  xanthine,  hypoxanthine, 
thymine,  and  p-lactic  acid.  The  autolysis  of  the  leucsemic  spleen  was  much 
more  extensive  than  the  normal. 

Among  the  constituents  of  the  spleen  the  deposit  rich  in  iron,  which 
consists  of  ferruginous  granules  or  conglomerate  masses  of  them,  and  which 
is  derived  from  a  transformation  of  the  red  blood-corpuscles,  is  of  special 
interest.  It  was  closely  studied  by  Nasse.  This  deposit  does  not  occur  to 
the  same  extent  in  the  spleen  of  all  animals.  It  is  found  especialh^  abun- 
dant in  the  spleen  of  the  horse.  Nasse  ^  on  analyzing  the  grains  (from  the 
si)leen  of  a  horse)  obtained  840-630  p.  m.  organic  and  160-370  p.  m.  inor- 
ganic substances.  These  last  consisted  of  566-726  p.  m.  Fe203,  205-388 
p.  m.  P2O5,  and  57  p.  m.  earths.  The  organic  substances  consisted  chiefly 
of  proteins  (660-800  p.  m.),  nuclein  (52  p.  m.  maximum),  a  yellow  color- 
ing-matter, extractive  bodies,  fat,  cholesterin,  and  lecithin. 

In  regard  to  the  mineral  constituents,  it  is  to  be  observed  that  in  compari- 
son with  sodium  and  phosphoric  acid  the  amount  of  potassium  and  chlorine 

'  Levene,  Zeitschr.  f.  physiol.  Chem.,  3";   Levene  and  Mandel,  ibid.,  45  and  -t". 
^  See  Chapter  XV  for  the  literature. 

'  Hedin  and  Rowland,  Zeitschr.  f.  physiol.  Chem.,  32,  and  Hedin,  Journ.  of  Physiol.  . 
30;   Leathes,  Journ.  of  Physiol.,  28;   Schumm,  Hofmeister's  Beitrage,  3  and  7. 
*  Maly's  Jahresber.,  19,  p.  315. 


274  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

is  ^mall.  The  amount  of  iron  in  new-born  and  3'oimg  animals  is  small 
(Lapicque,  Kruger,  and  Pernou),  in  adults  more  appreciable,  and  in  old 
animals  sometimes  very  coasiderable.  Nasse  found  nearly  50  p.  m.  iron 
in  the  dried  pulp  of  the  spleen  of  an  old  horse.  Guillemonat  and  La- 
picque 1  have  determined  the  iron  in  man.  They  find  no  regular  increase 
with  growth,  but  in  most  cases  0.17-0.39  p.  m.  (after  subtracting  the  blood- 
iron)  calculated  on  the  fresh  substance.  A  remarkably  high  amount  of 
iron  is  not  dependent  upon  old  age,  but  is  a  residue  from  chronic  diseases. 

The  quantitative  analyses  of  the  human  spleen  by  Oidtmann  ^  give  the 
following  results:  In  men  he  found  750-694  p.  m.  water  and  250-306  p.  m. 
solids.  In  that  of  a  woman  he  found  774.8  p.  m.  water  and  225.2  p.  m. 
solids.  The  quantity  of  inorganic  bodies  was  in  men  4.9-7.4  p.  m.,  and  in 
women  9.5  p.  m. 

In  regard  to  the  pathological  processes  going  on  in  the  spleen  we  must 
specially  recall  the  abundant  re-formation  of  leucocytes  in  leucaemia  and 
the  appearance  of  amyloid  substance  (see  page  69). 

The  physiological  functions  of  the  spleen  are  little  known,  with  the 
exception  of  its  importance  in  the  formation  of  leucocytes.  Some  consider 
the  spleen  as  an  organ  for  the  dissolution  of  the  red  blood-corpuscles,  and 
the  occurrence  of  the  above-mentioned  deposit  rich  in  iron  seems  to  con- 
firm this  view.  The  spleen  has  also  been  claimed  to  play  a  certain  part  in 
digestion.  This  organ  is  said  by  Schiff,  Herzen,  and  others  to  be  of 
importance  in  the  production  of  trypsin  in  the  pancreas.  The  investi- 
gations of  Herzen  seem  to  confirm  this  relation,  but  the  recent  work  of 
Prym^  has  made  the  assumption  doubtful. 

An  increase  in  the  quantity  of  uric  acid  eliminated  in  splenic  leucaemia 
has  been  observed  by  many  investigators  (see  Chapter  XV),  while  the 
reverse  has  been  observed  under  the  influence  of  quinine  in  large  doses, 
which  produces  an  enlargement  of  the  spleen.  These  facts  give  a  rather 
positive  proof  that  there  is  a  close  relationship  between  the  spleen  and 
the  formation  of  uric  acid.  This  relationship  has  been  studied  by  Horbac- 
ZEWSKi.  He  has  shown  that  when  the  spleen-pulp  and  blood  of  calves 
are  allowed  to  act  on  each  other,  under  certain  conditions  and  temperature, 
in  the  presence  of  air,  large  quantities  of  uric  acid  are  formed.  Under 
other  conditions  he  obtained  from  the  spleen-pulp  only  xanthine  bodies 
with  very  little  or  no  uric  acid.      Horbaczewski  ^  has  also  shown  that  the 

*  Lapicque,  ibid.,  20;  Lapicque  and  Guillemonat,  Compt.  rend,  de  soc.  biol.,  iS, 
and  Arch.,  de  Physiol.  (.5),  8;  Kriiger  and  Pernou,  Zeitsclir.  f.  Biologie,  27;  Nasse, 
cited  from  Hoppe-Seyler,  Physiol.  Chem.,  720. 

^  Cited  from  v.  Gorup-Besanez,  Lehrbuch,  4.  Aufl.,  p.  719. 

3  Schiff,  cited  by  Herzen,  Pfliiger's  Arch.,  30,  295,  308,  and  84,  and  Maly's  Jahr- 
esber.,  IS;   Prym,  Pfliiger's  Arch.,  lO-l  and  107;   see  also  Chapter  IX. 

*  Monatshefte  f.  Chem.,  10,  and  Wien.  Sitzungsber.  Math.  Nat.  Klasse,  100,  Abt,  3. 


THYROID   GLAXD.  275 

uric  acid  originates  from  the  nucleins  of  the  spleen,  which  yield  uric  acid 
and  xanthine  bodies  according  to  the  experimental  conditioas.  This 
behavior  is  explained  by  the  above-mentioned  investigations  of  Buriax, 
ScHiTTEXHELM,  JoxES,  and  others  on  the  enzj-motic  uric-acid  formation 
and  the  deamidization  of  the  purine  l^odies,  and  a  relationship  between  the 
spleen  and  uric-acid  formation  is  indisputable.  Still  we  cannot  say  that 
the  spleen  shows  a  special  relationship  to  the  uric-acid  formation  as  com- 
pared with  other  organs  (see  Chapter  XX). 

The  spleen  has  the  same  property  as  the  liver  of  retaining  foreign  bodies, 
metals  and  metalloids. 

The  Thyroid  Gland.  The  nature  of  the  different  protein  substances 
occurring  in  the  thj-roid  gland  has  not  been  sufficiently  studied,  but  at 
present,  through  the  researches  of  Oswald,  there  are  known  at  least  two 
bodies  which  are  constituents  of  the  so-called  secretion  of  the  glands.  One 
of  these,  iodothyreoglobulin,  behaves  like  a  globulin,  while  the  other  is  a 
nucleoproteid  (see  also  Gourlay^).  The  iodine  present  in  the  gland 
occurs  chiefly  in  the  first  body,  while  the  arsenic,  which  has  been  shown 
to  be  a  normal  constituent  by  Gautier  and  Bertraxd,^  seems  to  be  re- 
lated to  the  nuclein  substances. 

According  to  0sw^\ld  the  iodothyreoglobulin  occurs  only .  in  those 
glands  which  contain  colloid,  while  the  colloid-free  glands,  the  parenchyma- 
tous goitre,  and  the  glands  of  the  new-lDorn  contain  thyreoglobulin  free 
from  iodine.  The  thyreoglobuhn  first  becomes  iodized  into  iodothyreo- 
globulin on  passing  from  the  follicle-cells.  Besides  these  mentioned  bodies 
leucine,  xanthine,  hypoxanthine,  iodothyrine,  lactic  and  succinic  acids  occur 
in  the  thyreoidea.  Oidtivl^xx  ^  found  in  the  thyroid  gland  of  an  old 
woman  822.4  p.  m.  water.  176.6  p.  m.  organic  and  0.9  p.  m.  inorganic 
substances.  He  found  772.1  p.  m.  water,  223.5  p.  m.  organic  and  4.4 
p.  m.  inorganic  substances  in  an  infant  two  weeks  old. 

In  "struma  cystica"  Hoppe-Seyler  found  hardly  any  protein  in  the  smaller 
glandular  vessels,  but  an  excess  of  mucin,  while  n  the  larger  he  found  a  great 
deal  of  'protein,  70-80  p.  m.*  Cholesterin  is  regularly  found  in  such  cj'sts,  some- 
times in  such  large  quantities  that  the  entire  contents  form  a  thick  mass  of  cho- 
lesterin plates.  Crystals  of  calcium  oxalate  also  occur  frequently.  The  contents 
of  the  struma  cysts  are  sometimes  of  a  brown  color  due  to  decomposed  coloring- 
matter,  viethcemoglohin  (and  hsematin?).  Bile-coloring  matters  have  also  been 
found  in  such  cysts.  (In  regard  to  the  paralbumins  and  colloids  which  have  been 
foimd  in  struma  cysts  and  colloid  degeneration,  see  Chapter  XIII.) 

^  Gourlay,  Journ.  of  Physiol.,  16;  Oswald,  Zeitsclir.  f.  i^hysiol.  Chem..  32,  and 
Biochem.  Centralbl.,  1,  429. 

'Gautier,  Compt.  rend.,  129.  See  also  ibid.,  130,  131,  134,  135;  Bertrand,  ibid., 
134,  135. 

3 1.  c,  732. 

*  Physiol.  Chem.,  p.  721. 


276  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

Those  substances  which  bear  a  close  relationship  to  the  functions  of 
the  gland  seem  to  be  of  special  interest. 

The  complete  extirpation,  as  also  the  pathological  destruction,  of  the 
thyroid  gland  causes  great  disturbances,  ending  finally  in  death.  In  dogs, 
after  the  total  extripation,  a  disturbance  of  the  nervous  and  muscular  sys- 
tems occurs,  such  as  trembhng  and  con\adsions,  and  death  generally  super- 
venes shortly  after,  most  often  during  such  an  attack.^  In  human  beings 
different  disturbances  appear,  such  as  nervous  symptoms,  diminished  in- 
telligence, diyness  of  the  skin,  falhng  out  of  the  liair,  and,  on  the  whole, 
those  symptoms  which  are  included  under  the  name  cachexia  thyreopriva, 
and  death  follows  gradually.  Among  these  symptoms  must  be  mentioned 
the  peculiar  slimy  infiltration  and  exuberance  of  the  connective  tissue 
called  myxcedema.  It  has  been  proved  that  the  destructive  action  of  the 
removal  of  the  thyroid  can  be  counteracted  by  the  artificial  introduction 
of  extracts  of  the  thyroid  gland  into  the  body,  and  even  by  feeding  with 
the  substance  of  the  gland.  On  the  other  hand,  it  has  been  observed  on 
administering  too  large  quantities  of  gland  substance  that  threatening 
symptoms  and  disturbances  occur  in  man  as  well  as  in  animals.  From  a 
physiologico-chemical  standpoint  the  abnormally  increased  destruction  of 
body  protein,  occurring  on  continuous  feeding  witli  thyroid  preparations, 
is  of  the  greatest  importance. 

From  this  it  follows  that  the  glands  contain  specifically  active  sub- 
stances. It  is  impossible  for  the  present  to  state  anything  about  the  im- 
portance of  the  bases  found  by  certain  investigators,  such  as  S.  Frankel, 
Drechsel,  and  Kocher;^  these  bodies  have  not  been  characterized  suf- 
ficiently. It  seems  positively  proven  that  the  specifically  active  sub- 
stance is,  in  greater  part,  if  not  entirely,  as  first  show^n  b}'  Notkix,^  a  protein 
substance:  'Sotkis^s  thyreoproteid,  Oswald^s  thyreoglobulin.  This  does  not 
contradict  the  views  of  BAU^L\xx  and  Roos  that  the  active  substance  is 
iodothyrin,  as  this  is  produced  as  a  cleavage  product  from  the  iodothyreo- 
globulin. 

Iodothyrin  is  considered  by  Baumaxx,  who  first  showed  that  the  thjToid  con- 
tained iodine  and  who  with  Roos  *  showed  the  importance  of  this  substance  for  the 
physiological  activity  of  the  gland,  as  the  onh*  active  substance.  Iodoth\Tin  was 
obtained  by  Baumaxn  by  boiling  the  finely  di\'ided  gland  with  dilute  sulphuric 


'  The  divergent  statements  as  to  the  necessity  of  the  thyroid  gland  can  be  found  in 
H.  Munk,  Virchow's  Arch.,  150. 

^  Frankel,  Wien.  med.  Blatter,  1895  and  1896;  Drechsel  and  Kocher,  Centralbl. 
f.  Physiol.,  9,  705. 

^  Wien.  med.  Wochenschr.,  1895,  and  Virchow's  Arch.,  144,  SuppL,  224. 

*  In  regard  to  this  subject,  see  Baumann  and  Roos,  Zeitschr.  f.  physiol.  Chem.,  21 
and  22;  also  Baumann,  Mimch.  med.  Wochenschr.,  1896;  Baumann  and  Goldmann, 
ibid.;    Roos,  ibid.     An  extensive  review  of  the  literature  on  the  action  of  iodothyrin 


SUPRAREXAi.   CAPSULE.  277 

acid  as  an  amorphous,  brown  mass  nearly  insoluble  in  water  but  readily  soluble 
in  alkali  and  precipitated  again  by  the  addition  of  acid.  The  iodothjTin,  which 
is  not  a  unit  body,  has  a  variable  content  of  iodine  and  is  not  a  protein  substance. 

Thyreoglobulin  was  obtained  by  Oswald  from  the  water}-  extract  of 
the  gland  by  half  saturating  with  ammonium  sulphate.  It  has  the  proper- 
ties of  the  globulins  and  with  the  exception  of  the  iodine  content  it  has 
about  the  same  composition  as  the  proteins.  The  amount  of  iodine  varies: 
0.46  per  cent  in  pigs,  0.86  per  cent  in  oxen,  and  0.34  per  cent  in  man.  In 
young  animals,  whose  glands  contain  no  iodine,  the  thyreoglobulin  is 
iodine-free.  Thyreoglobulin  on  taking  up  iodine  is  converted  into  iodo- 
thyreoglobulin.  By  introducing  iodine  salts  the  iodine  content  of  the 
iodothyreoglobulin  can  be  raised  in  Uving  animals  and  thereby  also  the 
physiological  acti^-ity  increased  (Oswald).  The  amount  of  iodine  in  the 
gland  is  markedly  dependent  upon  the  food. 

According  to  Oswald  iodothyreoglobulin,  as  a  physiological  excitant  upon  the 
nervous  system,  has  a  regulating  action  upon  metabolism.  The  exclusion  of  this 
action,  after  destruction  or  extu-pation  of  the  gland,  explains,  according  to  Os- 
wald, the  injurious  results  produced  by  these  changes  upon  the  gland.  Accord- 
ing to  Blum  the  th\Toid  gland  removes  from  the  blood  a  poisonous  body,  the 
ihyreotoxalhumin,  and  makes  it  non-injurious  by  taking  up  iodine.  Kishi  ^  also 
believes  that  the  thjToid  gland  has  the  power  of  removing  poisons  from  the  blood. 
We  cannot  enter  further  into  this  and  other  related  questions. 

The  Suprarenal  Capsule.  Besides  proteins,  substances  of  the  connective 
tissue,  and  salts,  there  occur  in  the  suprarenal  capsule  inosite,  purine  bases, 
especially  xanthine  (Oker-Blom),  a  protagon-like  substance  (Orgler), 
relatively  considerable  lecithin  and  neurine,  and  glycerophosphoric  acid, 
which  are  probably  decomposition  products  of  the  lecithin.  The  older 
statements  on  the  occurrence  of  benzoic  acid,  hippuric  acid,  and  bile-acids 
are,  on  the  contrary-,  doubtful  and  are  not  substantiated  by  recent  inves- 
tigations (STADELiL\xx).  Older  investigators,  like  Vltlpiax  and  Arxold,^ 
have  found  in  the  medulla  a  chromogen  which  was  considered  to  be  con- 
nected with  the  abnormal  pigmentation  of  the  skin  in  Addison's  disease. 
This  chromogen,  which  is  transformed  by  air,  light,  alkahes,  iodine,  and 
other  bodies  into  a  red  pigment,  seems,  on  the  contrary-,  to  be  related  to 
the  substance  of  the  gland  producing  an  increase  in  the  blood-pressure. 

and  the  thyroid  preparations  can  be  found  in  Rocs,  Zeitschr.  f.  physiol.  Chem.,  22,  IS. 
In  regard  to  their  action  in  protein  destruction  and  metabolism,  see  F.  Voit,  Zeitschr. 
f.  Biologie,  35;  Schondorfif,  Pfliiger's  Arch.,  67,  and  Andersson  and  Bergman,  SkanJ. 
Arch.  f.  Physiol.,  S;   Magnus-Levy,  Zeitschr.  f.  klin.  Med.,  52. 

*  Kishi,  Virchow's  Arch.,  176.  A  summary  of  the  thyroid  hterature  for  the  last 
years  is  found  in  Maly's  Jahresber.,  24  and  25.  See  also  the  works  of  Blum  and  Oswald, 
cited  by  Oswald  in  Biochem.  Centralbl.,  1,  249. 

■  Oker-BIom,  Zeitschr.  f.  physiol.  Chem.,  2S;  Stadelmann,  ibid.,  IS,  wliich  also 
contains  the  literature  on  this  subject;   Orgler,  Salkowski's  Festschrift,  1904, 


278     CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

Adrenalin  (siiprarenin,  epinephrin).  That  the  watery  extract  of  the 
suprarenal  capsule  has  a  blood-pressure-raising  action  was  shown  by 
Oliver  and  Schafer,  Cybulski  and  Szymonowicz.^  The  substance  which 
is  here  active  was  formerly  called  sphygmogenin  and  has  also  other  actions 
besides  bringing  about  a  marked  increase  in  blood-pressure  by  the  strong 
contraction  of  the  muscles  of  the  periphery  vessels;  for  instance,  it  can  bring 
about  glycosuria.  This  body  has  been  chemically  investigated  by  several 
experimenters  and  has  received  different  names,  v.  FtJRTH  calls  it  supra- 
renin,  Abel  epinephrin,  and  Takamine  adrenalin.  This  last  name  seems 
to  be  the  most  generally  accepted  one.  The  chemical  constitution  of 
adrenalin  has  not  been  positively  decided  upon.  Aldrich  gives  the 
formula  C9H13NO3  for  adrenalin,  and  this  formula  has  been  accepted  as 
correct  by  a  large  number  of  investigators,  such  as  v.  Furth,  Jowett, 
Pauly,  Abderhalden  and  Bergell,  Bertrand,  Friedmann,  and  Stolz, 
basing  their  opinion  upon  their  own  researches.^  Abel  disputes  the  correct- 
ness of  this  formula  and  considers  adrenalin  as  a  hydrate  of  a  substance, 
C10H13NO3,  called  epinephrin  by  him,  hence  it  is  epinephrin  hydrate, 
CioHi3N03  +  §H20.  The  general  opinion  in  regard  to  the  constitution  of 
adrenalin  is  that  it  contains  a  pyrocatechin  complex,  three  OH  groups,  of 
Avhich  one  is  found  in  the  side-chain,  and  one  CH3NH  group.  The  formula 
(HO)2.C6H3.CH(OH).CH2.NH.CH3,  given  by  Pauly,  according  to  the  in- 
vestigations of  Friedmann,  can  be  accepted  as  correct.  Based  upon  these 
facts  it  has  been  possible  to  prepare  compounds  synthetically  whose  phy- 
siological action  was  more  or  less  similar  to  adrenalin,  namely,  by  starting 
from  pyrocatechin,  especially  by  treating  chloracetopyrocatechin  with 
ammonia,  alkylamines,  and  other  basic  bodies  (Stolz,  Meyer,  Friedmann, 
Dakin).3 

Adrenalin  is  soluble  in  water,  precipitated  by  ammonia,  and  thereby 
separates  as  crystals.  It  gives  an  emerald-green  color  with  ferric  chloride 
in  acid  solution  and  a  carmine-red  coloration  in  alkaline  solution.  It  re- 
duces Fehling's  solution  and  an  ammoniacal  silver  solution.  Epinephrin 
(Abel)  is  precipitated  by  several  alkaloid  reagents  and  gives  color  reac- 
tions with  JNIandelin's  alkaloid  reagent  and  with  permanganate  and  sul- 

'  Oliver  and  Schafer,  Proceed,  of  Physiol.  Soc,  London,  1895.  Further  literature 
on  the  function  of  the  suprarenal  capsule  may  be  found  in  Szymonowicz,  Pfliiger's 
Arch.,  64. 

^  The  literature  on  this  subject  may  be  found  in  v.  Fiirth,  Zeitschr.  f.  physiol.  Chem., 
23,  26,  29,  and  Wien.  Sitzungsber.  Math.  Nat.  Kl.,  112,  1903.  See  also  Abel,  Zeitschr.' 
f.  physiol.  Chem.,  2S;  .A.mer.  Journ.  of  Physiol.,  1899,  and  Tlie  Johns  Hopkins  Hospi- 
tal Bull.,  No.  76  (1897),  90  and  91  (1898),  120  and  12S  (1901),  131  and  132  (1902); 
Rer.  d.  d.  chem.  Gesellsch.,  36;  Abel  and  Taveau,  Journ.  of  Biol.  Chem.,  1,  and  Fried- 
mann, Hofmeister's  Beitrage.  6  and  8. 

*  Stolz,  Ber.  d.  d.  cliem.  Gesellsch.,  37;  Friedmann,  Hofmeister's  Beitrage,  6  and 
8;   Dakin,  Proc.  Roy.  Soc,  1905,  Ser.  B,  Vol.  76. 


ADRENALIN.  279 

phuric  acid.  On  this  point  the  conditions  are  not  quite  clear.  According 
to  Abel  the  crj'stalline  substance  (his  epineplirin  hydrate)  precipitated  by 
ammonia,  which  corresponds  to  the  adrenaUn  of  the  other  investigators, 
does  not  have  the  alkaloid  properties  of  epinephrin,  but  is  converted  by 
the  action  of  mineral  acids  into  epinephrin.  The  epinephrin  is  probably  a 
transformation  product  of  adrenalin.  Further  investigation  is  necessar}'- 
before  this  can  be  explained. 

The  glycosuria  first  observed  by  Blum  after  the  injection  of  the  extract 
of  the  suprarenal  capsule  is  due  to  an  action  of  the  adrenalin,  and  it  is 
hardly  possible  that  the  diastatic  enzyme  found  in  the  suprarenaJ  capsule 
by  Croftan  ^  takes  any  part  in  this  change. 

'  Blum,  Pfliiger's  Arch.,  90;   Croftan,  ibid.,  90. 


CHAPTER  VIII. 
THE  LIVER. 

The  liver,  which  is  the  largest  gland  of  the  body,  stands  in  close  rela- 
tionship to  the  blood-forming  glands.  The  importance  of  this  organ  for 
the  physiological  composition  of  the  blood  is  evident  from  the  fact  that  the 
blood  coming  from  the  digestive  tract,  laden  with  absorbed  bodies,  must 
circulate  through  the  liver  before  it  is  driven  by  the  heart  through  the 
different  organs  and  tissues.  It  has  been  proved,  at  least  for  the  carbo- 
hydrates, that  an  assimilation  of  the  absorbed  nutritive  substances  which 
are  brought  to  the  liver  by  the  blood  of  the  portal  vein  takes  place  in  this 
organ,  and  there  is  no  doubt  that  synthetical  processes  also  occur.  The 
occurrence  of  synthetical  processes  in  the  liver  has  been  positively  proved 
by  special  observations.  It  is  possible  that  in  the  liver  certain  ammonia 
combinations  are  converted  into  urea  or  uric  acid  (in  birds)  (see  Chapter 
XV),  while  certain  products  of  putrefaction  in  the  intestine,  such  as  phenols, 
may  be  converted  by  synthesis  into  ethereal  sulphuric  acids  by  the  liver 
(Pflijger  and  Kochs,  Embden  and  Glaessner),  probably  also  converted 
into  conjugated  glucuronic  acids  (Embden  i).  The  hver  has  also  the  prop- 
erty of  removing  and  retaining  heterogeneous  bodies  from  the  blood,  and 
this  is  not  only  true  of  metallic  salts,  which  are  often  removed  by  this 
organ,  but  also,  as  Schiff,  Heger,  and  others,  but  especially  Roger,  have 
shown,  the  alkaloids  are  retained,  and  are  probably  also  partially  decom- 
posed in  the  liver.  Toxines  are  also  withheld  by  the  liver,  and  hence  this 
organ  has  a  protective  action  against  poisons.^ 

Even  though  the  liver  is  of  assimilatory  importance  and  purifies  the 
blood  coming  from  the  digestive  tract,  it  is  at  the  same  time  a  secretory 
organ  which  eliminates  a  specific  secretion,  the  bile,  in  the  production  of 

^  Pfliiger  and  Kochs,  Pfliiger's  Arch.,  20  and  23;  Embden  and  Glaessner,  Hof- 
meister's  Beitrilge,  1;   Embden,  ibid.,  2. 

^  Roger,  Action  du  foie  sur  les  poisons  (Paris,  1887),  whicli  also  contains  the  older 
literature;  Bouchard,  Legons  sur  les  autointoxications  dans  les  maladies  (Paris,  1887); 
and  E.  Kotliar  in  Arch,  des  sciences  biologiques  de  St.  Petersbourg,  2.  See  also  de 
Vamossy,  Centralbl.  f.  Physiol.,  IS,  and  Rothberger,  Wien.  klin.  Wochenschr.,  1905, 
Rothborjrer  and  WintprKoro-,  Biochem.  Centralbl.,  4. 

280 


PROTEINS  OF  THE  LIVER.  281 

which  the  red  blood-corpuscles  are  destroyed,  or  at  least  one  of  their  con- 
stituents, the  haemoglobin.  It  is  generally  admitted  that  the  liver  acts 
contrari^\ise  during  foetal  life,  at  that  time  forming  the  red  blood-cor- 
puscles. 

There  is  no  doubt  that  the  chemical  operations  going  on  in  tliis  organ 
are  manifold  and  must  be  of  the  greatest  importance  for  the  organism. 
Our  knowledge  on  this  subject  has  been  essentially  advanced  by  the  recent 
investigations  on  the  enzymes  of  the  liver,  as  well  as  on  the  autolytic  pro- 
cesses in  tliis  organ,!  i^^^  nevertheless  it  must  be  admitted  that  our  knowl- 
edge of  the  character  and  extent  of  these  changes  is  still  small.  Among 
the  products  of  these  chemical  processes  there  are  two  which  are  especially 
important  and  must  be  treated  in  this  chapter,  namely,  the  glycogen  and 
the  bile.  Before  the  study  of  these  products  is  taken  up  a  short  discussion 
of  the  constituents  and  the  chemical  composition  of  the  Uver  is  necessary-. 

The  reaction  of  the  Uver-cells  is  alkaUne  towards  litmus  during  hfe,  but 
becomes  acid  after  death,  due  to  a  formation  of  lactic  acid,  chiefl}-  fer- 
mentation lactic  acid  and  other  organic  acids  (^IoRiSHnL\,  ^Iagxus-Le\'y2). 
A  coagulation  of  the  protoplasmic  proteins  in  the  cells  probably  takes 
place.  A  positive  difference  between  the  proteins  of  the  dead  and  the 
living,  non-coagulated  protoplasm  has  not  been  observed. 

The  proteins  of  the  liver  were  first  carefully  investigated  by  Plosz.  He 
found  in  the  water}-  extract  of  the  liver  an  albuminous  substance  w^hich 
coagulates  at  45°  C,  also  a  globulin  which  coagulates  at  75°  C,  a  nu.cleo- 
albumin  which  coagulates  at  70°  C,  and  lastly  a  protein  body  which  is 
nearly  related  to  the  coagulated  albumins  and  which  is  insoluble  in  dilute 
acids  or  alkalies  at  the  ordinarv^  temperature,  but  dissolves  on  the  applica- 
tion of  heat,  being  converted  into  an  albuminate.  Halliburton  ^  has 
found  two  globuUns  in  the  hver-cells,  one  of  which  coagulates  at  68-70° 
C,  and  the  other  at  45-50°  C.  He  also  found,  besides  traces  of  albumin, 
a  nucleoproteid  which  possessed  1.45  per  cent  phosphorus  and  a  coagula- 
tion-point of  60°  C.  PoHL  has  obtained  an  "organ  plasma"  by  extracting 
the  finely  di^ided  liver  which  had  pre\"iously  been  entirely  freed  from 
blood  by  washing  with  8  p.  m.  XaCl  solution,  in  which  he  was  able  to 
detect  a  globulin  having  a  low  coagulation  temperature.  The  ver}-  varia- 
ble phosphorus  content  (0.28-1.3  per  cent)  of  this  globuUn  as  well  as  the 
insolubility  of  the  precipitates  produced  by  Httle  acid  in  an  excess  of  acid 
and  in  neutral  salts  seem  to  indicate  that  we  have  here  a  mixture  which 
consists  chiefly  of  nucleoproteids  and  not  of  globulins.     The  nearly  com- 

•  See  especially  the  works  of  Jacoby,  Zeitschr.  f.  physiol.  Chem.,  30;  Conradi,  Hof- 
meister's  Reitrage,  1;    Magnus-Le\'y,  ihid.,  2. 

2  Morishima,  Arch.  f.  exp.  Path.  u.  Pharm.,  43;   Magnus-Le%'y,  1.  c. 

^  Plosz.  Pfl'iger's  Arch.,  7;  Halliburton,  Joarn.  of  Physiol.,  13,  Suppl.  1892. 


282  THE  LIVER. 

plete  digestibility  with  pepsin-liydrochloric  acid  does  not  contradict  this 
assumption,  because,  as  is  known,  nucleoproteids  may  on  digestion  yield 
no  residue  (see  Chapter  V).  It  is  also  impossible  to  state  anything 
positive  about  the  nature  of  the  liver-globulin  found  by  Dastre,i  hav- 
ing a  coagulation  temperature  of  56°.  The  proteins  extractable  from  the 
liver  without  modification  must  be  thoroughly  investigated. 

Besides  the  above-mentioned  proteins  which  are  very  soluble,  the  liver- 
cells  contain  large  quantities  of  difficultly  soluble  protein  bodies  (see 
Plosz).  The  liver  also  contains,  as  first  shown  by  St.  Zaleski  and  then 
substantiated  by  several  other  investigators,  ferruginous  proteins  of  dif- 
ferent kinds. 2  The  chief  portion  of  the  protein  substances  in  the  liver 
seems  to  consist  in  fact  of  ferruginous  nucleoproteids.  On  boiling  the 
liver  with  water,  such  a  nucleoproteid  or  perhaps  several  are  spht,  and  a 
solution  is  obtained  containing  a  nucleic-acid-rich  nucleoproteid  or  a  mix- 
ture of  these  which  are  precipitable  by  acids.  This  protein  or  protein 
mixture,  which  has  been  called  ferratin  by  Schmiedeberg,^  has  been  care- 
fully studied  by  Wohlgemuth.^  The  quantity  of  phosphorus  was  3.06 
per  cent.  As  cleavage  products  on  hydrolysis  he  found  Z- xylose,  the  four 
nuclein  bases,  and  also  arginine,  lysine  (and  histidine?),  tyrosine,  leucine, 
glycocoll,  alanine,  a-proline,  glutamic  acid,  aspartic  acid,  phenylalanine,  oxy- 
aminosuberic  acid,  and  oxydiaminosebacic  acid  (see  Chapter  II). 

The  yellow  or  brown  pigment  of  the  liver  has  been  little  studied.  Dastre 
and  Floresco  ^  differentiate  in  vertebrates  and  certain  invertebrates  between  a 
ferruginous  pigment  soluble  in  water,  ferrine,  and  a  pigment  soluble  in  chloro- 
form and  insoluble  in  water,  chlorochrome.  They  have  not  isolated  these  pigments 
in  a  pure  condition.  In  certain  invertebrates  chlorophyll  originating  from  the 
food  also  occurs  in  the  liver. 

The  fat  of  the  liver  occurs  partly  as  very  small  globules  and  partly 
(especially  in  nursing  children  and  sucking  animals,  as  also  after  food  rich 
in  fat)  as  rather  large  fat-drops.  The  occurrence  of  a  fatty  infiltration,  i.e., 
a  transportation  of  fat  to  the  liver,  may  not  only  be  produced  by  an  excess 
of  fat  in  the  food  (Noel-Paton),  but  also  by  a  migration  from  other  parts 
of  the  body  under  abnormal  conditions,  such  as  posioning  with  phosphorus, 
phlorhizin,   and    certain   other    bodies    (Leo,  Lebedeff,  Rosenfeld,   and 

*  Pohl,  Hofmeister's  Beitrage,  7;    Dastre,  Compt.  rend.  see.  biolog.,  58. 

2  St.  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  10,  486;  Weltering,  ibid.,  21;  Spitzer, 
Pfliiger's  Arch.,  67. 

^  Arch.  f.  exp.  Path.  u.  Pharm.,  33;   see  also  Vay,  Zeitschr.  f.  physiol.  Chem.,  20. 

*  Wohlgemuth,  Zeitschr.  f.  physiol.  Chem.,  37,  42,  and  44,  and  Ber.  d.  d.  chem. 
Gesellsch.,  37.  See  on  liver  nucleoproteids  also  Salkowski,  Berl.  klin.  Wochenschr., 
1895;'  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  19;  Blumenthal,  Zeitschr.  f.  klin.  Med., 
84. 

•Arch,  de  Physiol.  (5),  10. 


JECORIX.  283 

others  ^).  The  fatty  infiltration  occurring  in  poisoning  and  which  is  accom- 
panied ^\ith  degenerative  changes  in  the  cells  may  cause  a  diminution  in 
the  amount  of  protein  and  a  rise  in  the  water  content.  If  the  amount  of 
fat  in  the  liver  is  increased  by  an  infiltration,  the  water  decreases  corre- 
sponcUngly,  while  the  quantity  of  the  other  solids  remains  little  changed. 
Changes  of  such  a  kind  may  occur,  so  that,  because  of  the  opposition 
(Rosexfeld)  existing  between  glycogen  and  fat,  a  Uver  rich  in  fat  is  habit- 
ually poor  in  glycogen.  The  reverse  occurs  after  feeding  -^ith  carbohy- 
drr.te-rich  food,  namely,  the  liver  is  rich  in  glycogen  and  poor  in  fat. 

The  composition  of  the  liver-fat  not  onl}^  seems  to  var}-  in  different 
animals,  but  is  variable  ^^ith  changing  conditions.  Thus  Xoel-Paton 
found  that  the  hver-fat  in  man  and  several  animals  was  poorer  in  oleic  acid 
and  had  a  correspondingly  higher  melting-point  than  the  fat  from  the 
subcutaneous  connective  tissue,  wliile  Rosexfeld  ~  has  observed  the 
opposite  condition  on  feeding  dogs  with  mutton-fat. 

Lecithin  is  a  normal  constituent  of  the  hver,  and  amounts  to  about  23.5 
p.  m.  according  to  Xoel-Patox.^  In  starvation  the  lecithin,  according  to 
Xoel-Patox,  forms  the  greatest  part  of  the  ethereal  extract,  while  with 
food  rich  in  fat,  on  the  contrary-,  it  forms  the  smallest  part.  Cholesterin 
occurs  only  in  small  quantities.  The  ethereal  extract  also  contains  a 
protagon-hke  body,  jecorin. 

Jecorin  was  first  found  by  Drechsel  in  the  liver  of  horses,  and  also  in  the 
liver  of  a  dolphin,  and  later  by  Baldi  in  the  liver  and  spleen  of  other  animals,  in 
the  muscles  and  blood  of  the  horse,  and  in  the  human  brain.  It  contains  sul- 
phur and  phosphorus,  but  its  constitution  is  not  positively  known.  Jecorin  dis- 
solves in  ether,  but  is  precipitated  from  this  solution  by  alcohol.  It  reduces 
copper  oxide,  and  it  solidifies  after  boiling  with  alkalies  to  a  gelatinous  mass. 
^Iaxasse  has  detected  dextrose  as  osazone  in  the  carbohydrate  complex  of  jecorin. 
It  may  lead  to  errors  in  the  investigations  of  organs  or  tissues,  for  it  can  easily 
be  mistaken  for  lecithin  on  account  of  its  solubilities  and  because  it  contains 
phosphorus. 

The  statement  by  Bing  that  jecorin  is  a  combination  of  lecithin  and  dextrose 
does  not  follow  from  the  analyses  of  jecorin  thus  far  known.  Jecorin  contains 
sulphur,  even  as  much  as  2.75  per  cent,  and  also  the  relation  of  P:X  in  lecithin  is 
1:1,  while  in  jecorin  it  is  quite  different,  1:2  to  1:6. 

The  variable  composition  and  divergent  properties  of  the  jecorin  isolated  and 
analyzed  by  various  investigators  *  make  it  very  possible  that  jecorin  is  a  mixture 

'  .Voel-Paton,  Journ.  of  Physiol.,  19;  Leo,  Zeitschr.  f.  physiol.  Chem.,  9;  Lebedeff, 
Pfliiger's  Arch.,  31;  Athanasiu,  Pfluger's  Arch.,  74;  Taylor,  Journ.  of  Exp.  Med.,  4; 
Kraus  u.  Sommer,  Hofmeister's  Beitrage,  2;  Rosenfeld,  Zeitschr.  f.  klin.  Med.,  36. 
See  also  Rosenfeld,  Ergebnisse  der  Physiologic,  1,  Abt.  1,  and  Berl.  klin.  Wochenschr., 
1904;   Schwalbe,  Centralbl.  f.  Phy.siol.,  18,  p.  319. 

^  Cited  by  Lummert,  Pfhiger's  Arch.,  71.  In  regard  to  the  liver-fat  of  children,  see 
Thiemich,  Zeitschr.  f.  physiol.  Chem.,  26. 

^  1.  c.     See  also  Hefter,  Arch.  f.  exp.  Path.  u.  Pharm.,  28. 

^  Drechsel,  Ber.  d.  sachs.  Gesellsch.  d.  Wissensch.,  18S6,  p.  44,  and  Zeitschr.  f.  Biol- 
ogie,  33;  Baldi,  Arch.  f.  (Anat.  u.)  Ph>siol.,  1887,  Suppl.  100;  Manasse,  Zeitschr.  f. 
physiol.  Chem.,  20;  Bing,  Centralbl.  f.  Physiol.,  12,  and  Skand.  Arch.  f.  Physiol.,  9; 
Meinertz,  Zeitschr.  f.  physiol.  Chem.,  46;   Siegfried  and  Mark,  ibid 


284  THE  LIVER. 

of  several  suHstances,  among  which  perhaps  occurs  a  sulphurized  and  phosphor- 
ized  substance  (Sifgfried  and  Mark). 

Among  the  extractive  substances  besides  glycogen,  which  will  be  treated 
later,  rather  large  quantities  of  the  xanthine  bodies  occur.  Kossel  ^  found 
in  1000  parts  of  the  dried  substance  1.97  p.  m.  gimnine,  1.34  p.  m.  hypo- 
xanthine,  and  1.21  p.  m.  xanthine.  Adenine  is  also  contained  in  the  liver. 
In  addition  there  have  been  found  urea  and  uric  acid  (especially  in  birds), 
and  indeed  in  larger  quantities  than  in  the  blood,  paralactic  acid,  leucine,  and 
cystine.  In  pathological  cases  inosite  and  tyrosine  have  been  detected.  The 
occurrence  of  bile-coloring  matters  in  the  liver-cell  under  normal  conditions 
is  doubtful;  but  in  retention  of  the  bile  the  cells  may  absorb  the  coloring- 
matter  and  become  colored  thereby. 

A  large  number  of  enzymes  are  found  in  the  liver,  among  which  (besides 
the  cataloJies,  the  oxidases,  the  glycolytic  enzyme,  which  will  be  spoken  of 
later,  the  enzymes  taking  part  in  the  formation  of  uric  acid  and  destruction, 
of  uric  acid  (Chapter  XV),  the  arginase  which  forms  urea,  and  the  diastase 
acting  upon  glycogen)  we  must  mention  the  so-called  Zijmse  and  the  proteo- 
lytic enzyme.  The  liver  has  the  power  of  splitting  various  esters,  an  action 
which  has  been  recently  studied  by  Dakin,2  and  this  action  is  due  to  an 
enzyme  which  is  considered  as  a  lipase.  The  nature  of  this  lipase,  whose 
cleavage  action  upon  the  amyl  ester  of  salicylic  acid  was  first  observed  by 
Chanoz  and  Doyen,  has  been  closely  studied  by  Magnus,^  and  it  has  been 
shown  that  this  action  is  the  result  of  the  combined  action  of  two  sub- 
stances. The  lipase  solution  becomes  inactive  by  dialysis,  a  thermostable 
substance  soluble  in  absolute  alcohol  passing  into  the  diffusate,  and  this 
body  acts  as  a  co-enzyme,  making  the  solution  which  had  been  made  inac- 
tive by  dialysis  active  again. 

The  proteolytic  enzymes  of  the  liver  are  of  special  interest,  especially 
in  regard  to  the  study  of  the  autolysis  of  this  organ.  The  processes  in  the 
liver  in  phosphorus  poisoning  and  in  acute  yellow  atrophy  of  the  liver  are 
considered  as  an  intra  vitally  increased  autolysis.  In  these  cases  a  softening 
of  the  organ  takes  place,  and  proteoses,  mon-  and  diamino-acids,  and  other 
bodies  are  produced,  which  may  also  in  part  be  found  in  the  urine,  and 
although  they  may  not  all  be  derived  from  the  liver  (Neuberg  and  Rich- 
ter),  are  at  least  in  part  derived  from  this  organ. 

Wakeman*  has  found  in  phosphorus  poisoning  that  not  only  is  the 
quantity  of  nitrogen  markedly  diminished  in  the  liver  (of  dogs),  but  also 

*  Zeitschr.  f.  physiol.  Chem.,  8. 
2  Journ.  of  Physiol.,  30  and  32. 

'  Chanoz  and  Doyen,  Journ.  de  physiol.  et  de  path,  g^n^ral,  2;  Magnus,  Zeitschr.  f. 
physiol.  Chem..  42. 

*  Neuberg  and  Richter,  Deutsch.  Med.  Wochenschr.,  1904;  Wakeman,  Zeitschr.  f. 
physiol.  Chem.,  44. 


MINERAL  CONSTITUENTS  OF   THE   LIVER.  285 

that  the  quantity  of  nitrogen  of  the  hexone  bases  is  diminished,  and  that  the 
part  of  the  protein  molecule  richer  in  nitrogen  is  first  removed  and  elimi- 
nated under  these  conditions.  The  increased  consumption  of  glycogen  under 
the  above-mentioned  pathological  conditions  may  also  be  considered  as  an 
increased  autolysis. 

Besides  the  above-mentioned  organic  constituents  in  the  liver  we  must 
mention  the  glucothionic  acid  found  by  Mandel  and  Levene,  whose  rela- 
tionship to  the  carbohydrate  metabohsm  in  this  organ,  as  well  as  to  the 
nitrogenous  carbohydrate  found  by  Seegen  and  Neimann  ^  in  the  liver^ 
requires  further  investigation. 

The  mineral  bodies  of  the  liver  consist  of  phosphoric  acid,  potassium, 
sodium,  alkaline  earths,  and  chlorine.  The  potassium  is  in  excess  of  the 
sodium.  Iron  is  a  regular  constituent  of  the  liver,  but  it  occurs  in  very 
variable  amounts.  Bunge  has  found  0.01-0.355  p.  m.  iron  in  the  blood- 
free  Uver  of  young  cats  and  dogs.  This  was  calculated  on  the  liver  sub- 
stance freshly  washed  with  a  1  per  cent  NaCl  solution.  Calculated  on  10 
kilos  bodily  weight,  the  iron  in  the  liver  amounted  to  3.4-80.1  mg.  Recent 
determinations  of  the  quantity  of  iron  in  the  liver  of  the  rabbit,  dog,  hedge- 
hog, pig,  and  man  have  been  made  by  Guillemonat  and  Lapicque.  The 
variation  was  great  in  human  beings.  In  men  the  quantity  of  iron  in  the 
blood-free  liver  (blood-pigment  subtracted  in  the  calculation)  was  regularly 
more,  and  in  women  less,  than  0.20  p.  m.  (calculated  on  the  fresh  moist 
organ).  Above  0.5  p.  m.  is  considered  as  pathological.  According  to 
BiELFELD,2  who  also  finds  a  greater  iron  content  in  men,  this  difference 
appears  only  after  the  first  20-25  years.  At  this  age  (20-25  years)  the  iron 
content  is  smallest. 

The  quantity  of  iron  in  the  liver  can  be  increased  by  drugs  containing 
iron,  as  also  by  inorganic  iron  salts,  and  the  largest  deposition  of  iron  was 
observed  by  Novi^  after  the  hypodermic  injection  of  iron.  The  quantity 
of  iron  may  also  be  increased  by  an  abundant  destruction  of  red  blood- 
corpuscles,  which  will  result  from  the  injection  of  dissolved  haemoglobin,  in 
which  process  the  iron  combinations  derived  from  the  blood-pigments  in 
other  organs,  such  as  the  spleen  and  marrow,  also  seem  to  take  part.* 
A  destruction  of  blood-pigments,  with  a  splitting  off  of  compounds  rich 
in  iron,  seems  to  take  place  in  the  hver  in  the  formation  of  the  bile-pig- 
ments.    Even  in  invertebrates,  which  have  no  haemoglobin,  the  so-called 


'  Mandel  and  Levene,  Zeitsphr.  f.  physiol.  Chem.,  45;  Seegen,  Ceutralbl.  f.  Physiol., 
12  and  13,  with  Neimann,  Wiener  Sitzungsber.  Math.  Klasse,  112. 

^  Bunge,  Zeitschr.  f.  physiol.  Chem.,  17,  78;  Guillemonat  and  Lapicque,  Compt. 
rend,  de  soc.  biol.,  48,  and  Arch,  de  Physiol.  (5),  8;  Bielfeld,  Hofmeister's  Beitrage, 
2;   see  also  Schmey,  Zeitschr.  f.  physiol.  Chem.,  39. 

*  See  Centralbl.  d.  Physiol.,  16,  393. 

*  See  Lapicque,  Compt.  rend.,  124,  and  Schurig,  Arch.  f.  exp.  Path.  u.  Pharm.,  41. 


286  THE   LIVER. 

liver  is  rich  in  iron,  from  which  Dastre  and  Floresco  ^  concKicle  that  the 
quantity  of  iron  in  the  liver  of  invertebrates  is  entirel}'  independent  of  the 
decomposition  of  the  blood-pigment,  and  in  vertebrates  it  is  in  part  so. 
According  to  these  authors  the  Uver  has,  on  account  of  the  quantity  of 
iron,  a  specially  important  oxidizing  function,  which  they  call  the  "  fond  ion 
martiale"  of  the  liver. 

The  richness  in  iron  of  the  Uver  of  new-born  animals  is  of  special  inter- 
est— a  condition  which  was  shown  by  the  analyses  of  St.  Zaleski,  but  was 
especially  studied  by  Kruger  and  ]\Ieyer.  In  oxen  and  cows  they  found 
0.246-0.276  p.  m.  iron  (calculated  on  the  drj^  substance),  and  in  the  cow- 
fcetus  about  ten  times  as  much.  The  liver-cells  of  a  calf  a  week  old  contain 
about  seven  times  as  much  iron  as  the  adult  animal ;  the  quantity  sinks  in 
the  first  four  weeks  of  life,  when  it  reaches  about  the  same  amount  as  in  the 
adult.  Lapicque  -  has  also  found  that  in  rabbits  the  quantity  of  iron  in  the 
liver  steadily  diminishes  from  the  eighth  day  to  three  months  after  birth, 
namely,  from  10  to  0.4  p.  m.,  calculated  on  the  dry  substance.  "The  foetal 
liver-cells  bring  an  abundance  of  iron  into  the  world  to  be  used  up,  -v\dthin  a 
certain  time,  for  a  purjDOse  not  well  known."  A  part  of  the  iron  exists  as 
phosphate,  but  the  greater  part  is  in  combination  in  the  ferruginous  pro- 
tein bodies  (St.  Zaleski). 

The  quantity  of  calcium  oxide  in  the  fresh,  moist  liver  of  the  horse,  ox, 
and  pig,  according  to  Toyonaga,  amounts  to  0.148-0.193  p.  m.,  or  about 
the  same  as  in  the  human  liver.  The  amount  of  magnesium  oxide  was 
remarkably  high,  namely,  0.168,  0.198,  and  0.158  p.  m.,  in  the  Hvers  of  the 
horse,  ox,  and  pig  respectively.  Kjiuger^  has  found  the  quantity  of 
calcium  in  the  livers  of  adult  cattle  and  of  calves  to  be  respectively  0.71  p.  m. 
and  1.23  p.  m.  of  the  dried  substance.  In  the  foetus  of  the  cow  it  is  lower 
than  in  calves.  During  pregnancy  the  iron  and  calcium  in  the  foetus  are 
antagonistic;  that  is,  an  increase  in  the  quantity  of  calcium  in  the  liver 
causes  a  diminution  in  the  iron,  and  an  increase  in  the  iron  causes  a 
decrease  in  the  calcium.  Copper  seems  to  be  a  physiological  constituent, 
and  occurs  to  a  considerable  extent  in  cephalopods  (Henze).*  Foreign 
metals,  such  as  lead,  zinc,  and  others  (also  iron),  are  easily  taken  up  and 
combined  by  the  Hver  (Slowtzoff,  v.  Zeynek,  and  others  S). 

v.  Bibra  6  found  in  the  liver  of  a  young  man  who  had  suddenly  died 
762  p.  m.  water  and  238  p.  m.  solids,  consisting  of  25  p.  m.  fat,  152  p.  m. 


'  Arcfi.  de  Physiol.  (.')),  10. 

-St.  Zaleski,  1.  c;    Kruger  and  collaborators,  Zeitschr.  f.  Biologic,  2";    Lapicque, 
Maly's  Jahresber.,  20. 

^  Zeitschi.  f.  Biologic,  31;  Toyonaga,  Bull,  of  the  College  of  Agriculture,  Tokio,  6. 

*  Zeitschr.  f.  physiol.  Chem.,  33. 

*  Slowtzoff,  Hofmeister's  Beitriigc,  1;  v.  Zeynek,  see  Ccntralbl.  f.  Physiol.,  1,5. 
'  See  V.  Oorup-Besanez,  Lelirljucli  d.  physiol.  Chem.,  1.  Aufl.,  p.  711. 


GLYCOGEN  FORMATIOX.  287 

protein,  gelatine-forming  and  insoluble  substances,  and  61  p.  m.  extractive 
substances. 

The  quantitative  composition  of  the  liver  may  show  great  variation, 
depending  upon  the  kind  and  amount  of  the  food  supplied.  The  amount 
of  carbohydrate  (glycogen)  and  fat  may  var}'  considerably,  which  is  due  to 
the  fact  that  the  liver  is  a  storage-organ  for  these  bodies,  especiall)'-  for  the 
glycogen. 

Based  upon  special  experiments,  Seitz  ^  claims  that  the  hver  is  a  store- 
house also  for  protein.  In  experiments  on  hens  and  ducks  which  had  pre- 
viously been  starved,  he  fotmd  that  the  Hver  took  up  abimdant  protein  on 
feeding  meat  and  that  its  weight  as  compared  with  the  weight  after  star- 
vation was  doubled  or  quadrupled.  As  it  is  characteristic  of  storage  or 
reserve  bodies  that  their  amoimt  iu  the  storage-organs  on  feeding  \^ith  such 
bodies  strongly  increases  in  percentage,  it  is  remarkable  in  Seitz's  feeding 
experiments  that  the  percentage  of  protein  in  the  Uver  did  not  increase  but 
rather  diminished  sHghtly.  In  this  case  we  did  not  have  a  higher  percen- 
tage of  protein,  but  an  increase  in  the  weight  of  the  total  cell  mass  of  the 
organ,  probably  brought  about  by  increased  work  of  the  U^-er  due  to  the 
protein  feeding.  It  is  also  difficult  to  decide  as  to  how  far  in  these  experi- 
ments we  were  dealing  '^\ith  an  increase  in  the  number  or  the  size  of  the 
hver-celLs  or  with  a  deposition  of  reserve  protein  in  the  same  sense  as  of 
glycogen  or  excessive  fat. 

There  is  an  unanimous  beUef  that  the  hver  is  an  especially  important 
storage-organ  for  glycogen. 

Glycogen  and  its  Formation. 

Glycogen  was  first  discovered  by  Beexaed.  It  is  a  carbohydrate  closely 
related  to  the  starches  or  dextrins,  with  the  general  formula  (CeHioOslj.. 
Its  molecular  weight  is  unknown,  but  seems  to  be  verc'  large  (Gatix-Gru- 
ZEWSK.^  and  v.  Ivxaffl-Lexz^).  The  largest  quantities  are  found  in  the 
liver,  and  smaller  quantities  in  the  muscles  (Berxard.  Xasse).  It  is  found 
in  ver\-  small  quantities  in  nearly  all  tissues  of  the  animal  body.  Its  occur- 
rence in  lymphoid  cells,  blood,  and  pus  has  been  mentioned  in  a  pre\"ious 
chapter,  and  it  seems  to  be  a  regiilar  constituent  of  all  cells  capable  of 
development.  Glycogen  was  first  shown  to  exist  in  embrj-onic  tissues  by 
Berx.ard  and  Kuhxe.  and  it  seems  on  the  whole  to  l^e  a  constituent  of 
tissues  in  which  a  rapid  cell  formation  and  cell  development  is  taking 
place.  It  is  also  present  in  rapidly  forming  pathological  swelUngs  (Hoppe- 
Seyler).     Certain  animals,  as  certain  mussels  (Bizio').  tienia  and  ascarides 

iPfluger's  Arch..  111. 

^  Gatia-Gruzewska,  Pfliiger's  Arch.,  103;  v.  Ivnaffi-Lenz,  Zeitschr.  f.  physiol.  Chein., 
46. 


2S8  THE   LIVER. 

(Weinland  ^),   are  very   rich  in  glycogen.     Glycogen  also  occurs  in  the 
vegetable  kingdom,  especially  in  many  fungi. 

The  quantity  of  glycogen  in  the  liver,  as  also  in  the  muscles,  depends 
essentially  upon  the  food.  In  starvation  it  disappears  nearly  completely 
after  a  short  time,  but  more  rapidly  in  small  than  in  large  animals,  and  it 
disappears  earlier  from  the  liver  than  from  the  muscles.  After  partaking 
of  food,  especially  such  as  is  rich  in  carbohydrates,  the  liver  becomes  rich 
again  in  glycogen,  the  greatest  increment  occurring  14  to  16  hours  after 
eating  (Kxjlz).  The  quantity  of  liver-glycogen  may  amount  to  120-160 
p.  m.  after  partaking  of  large  quantities  of  carbohydrates,  and  in  dogs  which 
had  been  especially  fed  on  glycogen  Schondorff  and  Gatin-Gruzewska 
found  still  higher  results,  even  more  than  180  p.  m.  Ordinarily  it  is  con- 
siderably less,  namely,  12-30  to  40  p.  m.  According  to  Cremer  the  quan- 
tity of  glycogen  in  plants  (yeast-cells)  is,  as  in  animals,  dependent  upon  the 
food.  According  to  him  the  yeast-cells  contain  glycogen,  which  disappears 
from  the  cells  in  the  auto-fermentation  of  the  yeast,  but  reappears  on  the 
introduction  of  the  cells  into  a  sugar  solution. 

The  quantity  of  glycogen  of  the  liver  (and  also  of  the  muscles)  is  also  de- 
pendent upon  rest  and  activity,  because  during  rest,  as  in  hibernation,  it 
increases,  and  during  work  it  diminishes.  KtJLZ  has  shown  that  by  hard 
work  the  quantity  of  glycogen  in  the  liver  (of  dogs)  is  reduced  to  a  minimum 
in  a  few  hours.  The  muscle-glycogen  does  not  diminish  to  the  same  extent 
as  the  liver-glycogen.  Kulz,  Zuntz  and  Vogelius,  Frentzel,  and  others 
have  been  able  to  render  rabbits  and  frogs  glycogen-free  by  suitable  strych- 
nine poisoning.  The  same  result  is  produced  by  starvation  followed  by 
hard  work. 

Glycogen  forms  an  amorphous,  vvhite,  tasteless,  and  inodorous  powder. 
When  perfectly  pure  and  by  proper  alcohol  precipitation  it  can  be  obtained 
as  rods  or  prisms  which  look  like  crystals  (Gatin-Gruzewska).  It  gives 
an  opalescent  solution  with  water  which,  when  allowed  to  evaporate  on 
the  water-bath,  forms  a  pelUcle  over  the  surface  that  disappears  again  on 
cooling.  It  is  undecided  whether  we  have  here  a  true  solution  or  not. 
Like  other  colloids,  glycogen  in  water  under  the  influence  of  the  electric 
current  migrates  to  the  anode,  on  which  it  collects  (Gatin-Gruzewska). 
Its  aqueous  solution  is  dextrorotatory,  and  Huppert  found  it  to  be  (a)D  = 
+  196.63°.  Gatin-Gruzewska  has  recently  obtained  the  same  result  by 
using  a  perfectly  pure  solution  of  glycogen.  A  solution  of  glycogen,  es- 
pecially on  the  addition  of  NaCl,  is  colored  wine-red  by  iodine.     It  may 


'  Zeitschr.  f.  Biologic,  41.  The  extensive  literature  on  glycogen  may  be  found  in 
E.  Pfliiger,  Glykogen,  2.  Aufl.,  Bonn,  1905;  and  in  Cremer,  "  Physiol,  des  Glykogens,"  in 
Ergebnisse  der  Physiologic,  1,  Abt.  1.  In  the  following  pages  we  shall  refer  to  these 
works. 


PREPARATION  OF  GLYCOGEN.  289 

hold  cupric  hydrate  in  solution  in  alkaline  liquids,  but  does  not  reduce  it. 
A  solution  of  glycogen  in  water  is  not  precipitated  by  potassium-mercuric 
iodide  and  hydrochloric  acid,  but  is  precipitated  by  alcohol  (on  the  addition 
of  NaCl  when  necessarjO  or  ammoniacal  basic  lead  acetate.  An  aqueous 
solution  of  glycogen  made  alkaline  with  caustic  potash  (15  per  cent  KOH) 
is  completely  precipitated  by  an  equal  volume  of  96  per  cent  alcohol.  Tan- 
nic acid  also  precipitates  glycogen.  It  gives  a  white  granular  precipitate  of 
benzoyl  glycogen  with  benzoyl  chloride  and  caustic  soda.  Glycogen  is 
completely  precipitated  by  saturating  its  solution  at  ordinary  tempera- 
tures with  magnesium  or  ammonium  sulphate.  It  is  not  precipitated  by 
sodium  chloride  or  by  half  saturation  with  ammonium  sulphate  (Nasse,  Neu- 
MEiSTER,  Halliburton,  Young  i).  On  boiling  with  dilute  caustic  potash 
(1-2  per  cent)  the  glycogen  may  be  more  or  less  changed,  especially  if  it 
has  been  previously  exposed  to  the  action  of  acid  or  of  BrDcke's  reagent 
(see  below)  (PFLtJCER).  On  boiling  with  stronger  caustic  potash  (even  of 
36  per  cent)  it  is  not  injured  (Pflijger).  By  diastatic  enzymes  glycogen 
is  converted  into  maltose  or  dextrose,  depending  upon  the  nature  of  the 
enzyme.  It  is  transformed  into  dextrose  by  dilute  mineral  acids.  Ac- 
cording to  Tebb^  various  dextrins  appear  as  intermediary  steps  in  the 
saccharification  of  glycogen,  depending  on  whether  the  hydrolysis  is  caused 
by  mineral  acids  or  enzymes.  The  question  whether  the  glycogen  from 
various  animals  and  different  organs  is  the  same  in  this  regard  has  not  been 
sufficiently  investigated.  Nor  has  it  been  decided  whether  all  the  glycogen 
in  the  liver  occurs  as  such  or  whether  it  is  in  part  combined  with  protein 
(Pflijger-Nerking)  .  The  recent  investigations  of  Loeschcke  ^  have 
shown  that  we  have  no  positive  reasons  for  this  assumption. 

The  preparation  of  pure  glycogen  (most  easily  from  the  liver)  is  generalh^ 
performed  by  the  method  suggested  by  Brucke,  of  which  the  main  points 
are  the  following:  Immediately  after  the  death  of  the  animal  the  liver  is 
thrown  into  boiling  water,  then  finely  divided  and  boiled  several  times  with 
fresh  water.  The  filtered  extract  is  now  sufficiently  concentrated,  allowed 
to  cool,  and  the  proteins  removed  by  alternately  adding  potassium-mercuric 
iodide  and  hydrochloric  acid.  The  glycogen  is  precipitated  from  the 
filtered  liquid  by  the  addition  of  alcohol  until  the  liquid  contains  60  vols, 
per  cent.  By  repeating  this  and  precipitating  the  glycogen  several  times 
from  its  alkaline  and  acetic-acid  solution  it  is  purified  on  the  filter  by  wash- 
ing first  ^vith  60  per  cent  and  then  ^dth  95  per  cent  alcohol,  then  treating 
vnih  ether  and  drvdng  over  sulphuric  acid.  It  is  always  contaminated 
with  mineral  substances.  To  be  able  to  extract  the  glycogen  from  the 
liver  or,  especially,  from  muscles  and  other  tissues  completely,  which  is 
essential  in  a  quantitative  estimation,  these  parts  must  first  be  warmed 

'  Young,  Journ.  of  Physiol.,  22,  citing  the  other  investigators. 
^  Journ.  of  Physiol.,  22. 
3  Pfliiger's  Arch.,  102. 


290  THE  LIVER. 

for  two  hours  with  strong  caustic  potash  (30  per  cent)  on  the  water-bath. 
As  the  glycogen  changes  in  this  purification,  according  to  Brucke,  it  is 
better,  for  quantitative  determinations  of  glycogen,  to  precipitate  it  directly 
from  the  alkahne  solution  by  alcohol  (Pfluger  ^). 

The  quantitative  estimation  is  best  performed  according  to  PflIjger's 
method,  which  is  based  upon  the  following:  100  grams  of  the  finely  divided 
organ  and  100  c.c.  of  60  per  cent  caustic-potash  solution  are  heated  on  the 
water-bath  for  two  hours.  After  evaporating  the  water  to  400  c.c.  it  is 
filtered  through  glass  wool  and  the  glycogen  precipitated  from  100  c.c.  of 
the  filtrate  by  100  c.c.  of  96  per  cent  alcohol.  The  glycogen  is  washed  on 
the  filter  first  with  dilute  alkali  and  alcohol  and  then  with  alcohol  alone. 
It  is  then  dissolved  in  water,  exactly  neutralized,  treated  with  25  c.c. 
hydrochloric  acid  (1.19  sp.  gr.)  and  water  added  to  500  c.c,  when  the 
amount  of  HCl  will  be  2.2  per  cent.  On  heating  for  three  hours  the  glycogen 
will  have  been  converted  into  dextrose,  whose  quantity  can  be  determined 
according  to  Allihn-Pfluger's  method  by  reduction  of  an  alkaline  copper 
solution  and  weighing  the  cuprous  oxide.  As  a  control  the  weighed  cuprous 
oxide  is  dissolved  in  nitric  acid  and  the  copper  titrated  according  to  Vol- 
hard's  method.  In  regard  to  the  detailed  steps,  which  must  be  exactly 
observed,  compare  Pfluger's  original  work.  Other  methods  of  esti- 
mating glycogen,  such  as  those  of  BRiJCKE-KiJLZ,  Pavy,  and  Austin,  are 
descrilDed  in  PFLtJGER's  Archiv,  96.  See  also  the  new  method  as  suggested 
by  Salkowski  and  the  short  quantitative  analysis  of  glycogen  by  Pflijger.^ 

Numerous  investigators  have  endeavored  to  determine  the  origin  of 
glycogen  in  the  animal  body.  It  is  positively  established  by  the  unanimous 
observations  of  many  investigators  ^  that  the  varieties  of  sugars  and  their 
anhydrides,  dextrins  and  starches,  have  the  property  of  increasing  the  quan- 
tity of  glycogen  in  the  body.  The  action  of  inulin  seems  to  be  somewhat 
uncertain."*  The  statements  are  questioned  in  regard  to  the  action  of  the 
pentoses.  Cremer  found  in  rabbits  and  hens  that  various  pentoses,  such 
as  rhamnose,  xylose,  and  arabinose,  have  a  positive  influence  on  the  gly- 
cogen formation,  and  Salkowski  obtained  the  same  result  on  feeding 
Z-arabinose.  Frentzel  fdund,  on  the  contrar}^,  no  glycogen  formation  on 
feeding  xylose  to  a  rabbit  which  had  previously  been  made  glycogen-free 
by  strychnine  poisoning,  and  Neuberg  and  Wohlgemuth  ^  obtained  simi- 
lar negative  results  on  feeding  rabbits  with  d-  and  r-aral)inose. 

The  hexoses,  and  the  carbohydrates  derived  therefrom,  do  not  all  possess 
the  ability  of  forming  or  accumulating  glycogen  to  the  same  exteijt.     Thus 


'  See  also  the  method  suggested  by  Gautier,  Compt.  rend.,  129. 

2  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  36;  Pfluger,  Pfliiger's  Arch.,  103. 

^  In  reference  to  the  literature  on  this  subject,  see  E.  Kiilz,  Pfliiger's  Arch.,  24,  an  1 
Ludwig-Festschrift,  1891;  also  the  cited  works  of  Pfliiger  and  Cremer,  foot-note  1,  . 
288. 

*  See  Miura,  Zeitschr.  f.  Biologic,  32,  and  Nakaseko,  Amer.  Journ.  of  Physiol.,  4. 

*  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  32;  Neuberg  and  Wohlgemuth,  ibid.,  35. 
See  also  Pfliiger,  1.  c,  and  Cremer,  1.  c. 


GLYCOGEN   FORMATION.  231 

C  VoiT  1  and  his  pupils  have  shown  that  dextrose  has  a  more  powerful 
action  than  cane-sugar,  while  milk-sugar  is  less  active  (in  rabbits  and  hens) 
than  dextrose,  le\'ulose,  cane-sugar,  or  maltose.  The  following  substances 
when  introduced  into  the  body  also  increase  the  quantity  of  glycogen  ia 
the  liver:  glycerine,  gelatine,  arbutin,  and  like^\ise,  according  to  the  investi- 
gations of  KtJLZ,  erythrite,  quercite,  dulcite,  mannite,  inosite,  ethylene  and 
projyylene  glycol,  glucuronic  anhydride,  saccharic  acid,  mucic  acid,  sodium 
tartrate,  saccharine,  isosaccharine,  and  urea.  Ammonium  carbonate,  glycocoll^ 
and  asparagine  may  similarly,  according  to  Rohmann,  cause  an  increase  in 
the  amount  of  glycogen  in  the  liver.  According  to  Xebelthau  other 
ammonium  salts  and  some  of  the  amides,  as  well  as  certain  narcotics,  hyp- 
notics, and  antipyretics,  produce  an  increase  in  the  glycogen  of  the  Uver, 
This  action  of  the  antipyretics  (especially  antipyrine)  had  been  shown  by 
Lepine  and  Porteret.^ 

Pfluger  has  conclusively  shown  that  we  have  no  positive  proofs  as  to 
the  action  of  these  various  bodies  as  glycogen-formers.  That  glycerine  may 
in  a  positive  sense  influence  the  amount  of  glycogen  in  the  liver  is  not  to 
be  doubted  from  the  experiments  of  Weiss  and  Luchsinger  on  glycogen 
formation,  which  \<\\\  be  mentioned  in  connection  with  the  experiments  on 
the  relationship  of  glycerine  to  the  sugar  formation. 

The  fats,  according  to  Bouchard  and  Desgrez,  increase  the  glycogen 
content  of  the  muscles  but  not  of  the  liver,  and,  according  to  Coua'reur,^ 
the  glycogen  is  increased  at  the  expense  of  the  fat  in  the  silkworm  larva  as 
it  changes  into  a  chrs'saUs.  In  general  it  is  believed  that  fat  does  not  in- 
crease the  amount  of  glycogen  in  the  liver  or  in  the  animal  bod}-,  although 
a  carbohydrate  formation  from  glycerine,  but  not  a  glycogen  formation,  is 
probable.  Pfluger  explains  this  by  the  fact  that  the  extent  of  fat  metab- 
olism is  not  dependent  upon  the  quantity  of  fat  supplied,  but  upon  the 
amount  of  fat  required  conditioned  by  work.  If  more  fat  is  supplied,  then 
it  is  not  destroN^ed,  but  is  stored  up.  Even  when  sugar  is  continuously 
formed  from  the  fat  in  metabolism  this  is  immediately  burned  and  does 
not  yield  any  material  for  the  formation  of  the  reserve  substance  glycogen. 

The  views  in  regard  to  the  influence  of  the  proteins  are  somewhat  con- 
tradictor}-. From  several  investigations  the  conclusion  has  been  drawn 
that  the  proteins  cause  an  increase  in  the  glycogen  of  the  liver.  Amongst 
these  investigations  must  be  included  certain  feeding  experiments  with 
boiled  beef  (Xauxyx)  or  blood-fibrin  (v.  ^Ierixg),  and  especially  the  very 
careful  experiments  made  by  E.  Kulz  on  hens,  with  pure  protems.  .'^uch  as 


'  Zeitschr.  f.  Biologie,  2S. 

-  Rohmann,  Pfliiger's  Arch.,  39;  Nebelthau,  Zeitschr.  f.  Biologie,  28;  Lepine  ami  Por- 
teretjCompt.  rend.,  107. 

^  Bouchard  et  Desgrez,  Compt.  rend.,  130;  Couvreur,  Compt.  rend,  de  soc.  biol.,  47. 


292  THE   LIVER. 

casein,  seralbumin,  and  ovalbumin.  The  value  of  these  experiments  is 
disputed  by  Pflijger,  and  as  a  direct  proof  against  the  formation  of  gly- 
cogen from  protein  he  refers  to  Schondorff's  investigations  when  feeding 
carbohydrate-free  protein  (casein)  to  frogs  without  finding  the  least  in- 
crease in  the  total  glycogen.  Later  Blumenthal  and  Wohlgemuth 
arrived  at  similar  results.  They  found  no  glycogen  accumulation  in  frogs 
after  feeding  with  casein  or  gelatine,  but  did  find  it  after  feeding  with  oval- 
bumin, which  contains  a  carbohydrate  group.  On  the  contrary,  Bendix 
was  able  to  show  an  increase  in  the  glycogen  in  dogs  by  feeding  casein  and 
gelatine,  as  well  as  ovalbumin,  and  in  fact  a  greater  increase  by  casein  than 
by  ovalbumin.  Stookey  ^  arrived  at  similar  results  in  hens  as  he  found 
a  glycogen  formation  after  feeding  casein,  while  he  obtained  no  posi- 
tive results  after  feeding  glucoproteids.  It  seems  as  if  the  conditions  in 
cold-blooded  animals  were  different  from  those  in  warm-blooded  ones. 
According  to  Pflijger,  the  experiments  of  Bendix  are  not  conclusive,  and 
he  doubts  the  formation  of  glycogen  from  protein.  He  claims  it  is  only 
formed  from  carbohydrates  or  from  the  carbohydrate  complex  of  the 
glucoproteids. 

]Many  investigators  are  still  of  the  opinion  that  an  increase  in  the  gly- 
cogen of  the  liver  as  well  as  of  other  organs  can  be  brought  about  by  feeding 
animals  with  carbohydrate-free  proteins. 

If  the  question  is  raised  as  to  the  action  of  the  various  bodies  on  the 
accumulation  of  glycogen  in  the  liver,  it  must  be  recalled  that  a  formation 
of  glycogen  takes  place  in  this  organ,  as  well  as  a  consumption  of  the  same. 
An  accumulation  of  glycogen  may  be  caused  by  an  increased  formation  of 
glycogen,  but  also  by  a  diminished  consumption,  or  by  both. 

It  is  not  known  how  the  various  bodies  above  mentioned  act  in  this 
regard.  Certain  of  them  probably  have  a  retarding  action  on  the  transfor- 
mation of  glycogen  in  the  liver,  while  others  perhaps  are  more  combustible 
and  in  this  way  protect  the  glycogen.  Some  probably  excite  the  liver-cells 
to  a  more  active  glycogen  formation,  while  others  yield  material  from  which 
the  glycogen  is  formed  and  are  glycogen-formcrs  in  the  strictest  sense  of  the 
word.  The  knowledge  of  these  last-mentioned  bodies  is  of  the  greatest 
importance  in  the  question  as  to  the  origin  of  glycogen  in  the  animal  body, 
and  the  chief  interest  attaches  itself  to  the  question:  To  what  extent  are 
the  two  chief  groups  of  food,  the  proteins  and  carbohydrates,  glycogen- 
formers? 

The  great  importance  of  the  carbohydrates  in  the  formation  of  glycogen 
lias  given  rise  to  the  opinion  that  the  glycogen  in  the  liver  is  produced  from 

'  Schondorff,  Pfliiger's  Arch.,  82  and  88;  Blumenthal  and  Wohlgemuth,  Berl.  klin, 
Wochenschr.,  1901 ;  Bendix,  Zeitschr.  f.  physiol.  Chem.,  32  and  34;  Stookey,  Amer. 
Journ.  of  Physiol.,  9. 


GLYCOGEN  FORMATION.  293 

sugar  by  a  synthesis  in  which  water  separates  with  the  formation  of  an 
anhydride  (Luchsixger  and  others).  This  theor}^  {anhydride  theory)  has 
found  opponents  because  it  neither  explains  the  formation  of  glj^cogen 
from  such  bodies  as  proteins,  carbohydrates,  gelatine,  and  others,  nor  the 
circumstance  that  the  glycogen  is  always  the  same  independent  of  the 
properties  of  the  carbohydrate  introduced,  whether  it  is  dextrogyrate  or 
levogyrate.  It  used  to  be  the  opinion  of  many  investigators  that  all  gly- 
cogen is  formed  from  protein,  and  that  this  splits  into  two  parts,  one  con- 
taining nitrogen  and  the  other  being  free  from  nitrogen:  the  latter  is  the 
glycogen.  According  to  these  views,  the  carbohydrates  act  only  in  that 
they  spare  the  protein  and  the  glycogen  produced  therefrom  {sparing 
theory  of  Weiss,  Wolffberg,  and  others  i). 

In  opposition  to  this  theory  C.  and  E.  Voit  and  their  pupils  have  shown 
that  the  carbohydrates  are  "true"  glycogen-formers.  After  partaking  of 
large  quantities  of  carbohydrates  the  amount  of  glycogen  stored  up  in  the 
body  is  sometimes  so  great  that  it  cannot  be  covered  by  the  proteids  de- 
composed during  the  same  time,  and  in  these  cases  a  glycogen  formation 
from  the  carbohydrates  must  be  admitted.  According  to  Cremer  only  the 
fermentable  sugars  of  the  six  carbon  series  or  their  di-  and  polysaccharides 
are  true  glycogen-formers.  For  the  present,  only  dextrose,  levulose,  galac- 
tose (Weinland^)^  and  perhaps  also  rf-mannose  (Cremer)  are  designated 
as  true  glycogen-formers.  Other  monosaccharides  may  indeed,  according 
to  Cremer,  influence  the  formation  of  glycogen,  but  they  are  not  converted 
into  glycogen  and  hence  are  called  only  pseudoglycogen-formers. 

The  poly-  and  disaccharides  may,  after  a  cleavage  into  the  correspond- 
ing fermentable  monosaccharides,  serve  as  glycogen-formers.  This  is  true 
for  at  least  cane-sugar  and  milk-sugar,  which  must  first  be  inverted  in  the 
intestine.  These  two  varieties  of  sugar,  therefore,  cannot,  like  dextrose  and 
le\'ulose,  serve  as  glycogen-formers  after  subcutaneous  injection,  but  re- 
appear almost  entirely  in  the  urine  (Dastre,  Fr.  Voit).  ^laltose,  which  is 
inverted  by  an  enzyme  present  in  the  blood,  passes  only  to  a  slight  extent 
into  the  urine  (Dastre  and  Bourquelot  and  others),  and  it  can,  like  the 
monosaccharides,  even  after  subcutaneous  injection,  be  used  in  the  forma- 
tion of  glycogen  (Fr.  Voit  3). 

After  Pavy  ^  showed  the  glucoproteid  nature  of  ovalbumin  and,  as  dis- 

'  In  regard  to  these  two  theoiies,  see  especially  Wolffberg,  Zeitschr.  f.  Biologie,  KJ. 

2  E.  Voit,  Zeitschr.  f.  Biologie,  25,  543,  and  C.  Voit,  ibid.,  28.  See  also  Kausch 
and  Socin,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Weinland,  Zeitschr.  f.  Biolog'e,  -10  and 
38;   Cremer,  ibid.,  42,  and  Ergebnisse  der  Physiol.,  1. 

'  Dastre,  Arch,  de  Physiol.  (5),  3,  1891;  Dastre  and  Bourquelot,  Compt.  rend.,  98; 
Fritz  Voit,  Verhandl.  d.  Gesellsch.  f.  Morph.  u.  Physiol,  in  Miinchen,  1896,  and  Deutsch. 
Arch,  f  klin.  Med.,  58. 

*The  Physiology  of  the  Carbohydrates,  London,  1891. 


294  THE  LIVER. 

cusssd  later,  that  glucosamine  could  be  split  off  from  ovalbumin  as  well  as 
from  certain  other  protein  substances  (see  Chapter  II),  the  question  arose 
whether  the  amino-sugar  could  serve  in  the  formation  of  glycogen.  The 
investigations  carried  out  in  this  direction  by  Fabian,  Fraxkel  and  Offer, 
Cathcart  and  Bial,^  have  shown  that  the  glucosamine  introduced  into  the 
organism  is  in  part  eliminated  unchanged  in  the  urine  and  has  no  glycogen- 
forming  action.  No  definite  conclusions  can  be  drawn  from  this  on  the 
beha\-ior  of  the  carbohydrate  groups  wliich  exist  not  as  free  groups  but 
combined  wth  the  protein  molecules. 

Whether  or  not,  or  to  what  extent,  the  glucoproteids  take  part  in  the 
sugar  or  glycogen  formation  in  the  animal  body  is  difficult  to  answer  for 
the  present,  as  but  little  is  known  of  the  quantity  of  these  substances  in 
the  body  and  our  knowledge  of  the  amount  of  carbohydrate  which  can  be 
split  off  from  the  various  protein  substances  is  also  ver}'  meagre. 

From  the  weight  of  the  various  organs  and  the  relationship  of  the  weight 
of  the  organs  to  the  total  weight  of  the  body,  as  well  as  from  the  qualitative 
and  quantitative  composition  of  the  various  organs  as  far  as  known  for  the 
present,  we  can  calculate  the  carbohydrates  of  the  body  (excluding  the  gly- 
cogen), although  the  results  ma}-  not  be  exact,  but  no  doubt  are  too  high 
or  at  least  are  not  too  low.  These  calculations  of  Hammarsten  for  man 
arid  dogs  have  shown  that  in  the  nucleoproteids,  glucoproteids,  and  other 
substances  which  are  not  sugar  nor  glycogen,  but  for  the  sake  of  brevity 
are  called  glucosides,  the  maximum  carbohydrate  supply  of  the  body  is  5 
grams  per  1  kilo  of  body-weight. 

If  the  proteins  are  to  be  counted  among  those  bodies  which  can  increase 
the  glycogen  of  the  body,  then  we  must  ask  the  question:  Do  the  proteins 
act  only  indirectly  as  pseudoglycogen-formers  or  are  they  direct  glycogen- 
formers  which  can  serve  as  material  for  the  formation  of  glycogen  or  sugar? 
This  question  stands  in  close  relationsliip  to  the  sugar  formation  and  sugar 
elimination  in  the  various  forms  of  glycosuria  and  will  be  discussed  best 
below  in  connection  udth  the  question  of  diabetes. 

Like  the  carbohydrates  in  general,  glycogen  has  without  any  doubt  a 
great  importance  in  the  formation  of  heat  and  development  of  energ}'  in 
the  animal  body.  The  possibility  of  the  formation  of  fat  from  glycogen 
cannot  be  denied.^  Glycogen  is  generally  considered  as  reserve  food 
accumulated  in  the  liver  and  formed  in  the  liver-cells.  Where  does  the 
glycogen  existing  in  the  other  organs,  such  as  the  muscles,  originate?  Is 
the  glycogen  of  the  muscles  formed  on  the  spot  or  is  it  transmitted  to  the 
muscles  by  the  blood?    These  questions  cannot  yet  be  answered  with  posi- 

'  Fabian,  Zeitschr.  f.  physiol.  Chem.,  2";  Frankel  and  Offer,  Centralbl.  f.  Physiol., 
13;   Cathcart,  Zeitschr.  f.  physiol.  Chem.,  39;   Bial,  Berl.  klin.  Woclienschr.,  1905. 
^  See  especially  Noel-Paton,  Journ.  of  Physiol.,  19. 


VITAL  SUGAR   FORMATION.  295 

tiveness,  and  the  investigations  on  this  subject  l)y  different  experimenters 
have  given  contradictor}^  results.  The  experiments  of  KiJLz/  in  which  he 
studied  the  glycogen  formation  by  passing  blood  containing  cane-sugar 
through  the  muscle,  have  led  to  no  conclusive  results.  Still  the  formation 
of  glycogen  from  sugar  in  the  muscles  is  probable.  There  is  no  doubt  that 
glycogen  is  formed  in  the  muscles  during  embryonic  life. 

If  it  is  true  that  the  blood  and  lymph  contain  a  diastatic  enzyme  which 
transforms  glycogen  into  sugar,  and  also  that  the  glycogen  regularly  occurs 
in  the  form-elements  and  is  not  dissolved  in  the  fluids,  it  seems  probable 
that  the  glycogen  in  solution  is  not  transmitted  by  the  blood  to  the  organs, 
but  perhaps  more  likely,  if  the  leucocytes  do  not  act  as  carriers,  it  is  formed 
on  the  spot  from  the  sugar.^  The  glycogen  formation  seems  to  be  a  general 
function  Cf  the  cells.  In  adults,  the  liver,  which  is  very  rich  in  cells,  has 
the  property,  on  account  of  its  anatomical  position,  of  transforming  large 
quantities  of  sugar  into  glycogen. 

The  question  now  arises  whether  there  is  any  foundation  for  the  state- 
ment that  the  liver-glycogen  is  transformed  into  sugar. 

As  first  shown  by  Bernard  and  redemonstrated  by  many  investigators, 
the  glycogen  in  a  dead  hver  is  gradually  changed  into  sugar,  and  this  sugar 
formation  is  caused,  as  Bernard  supposed  and  Artiius  and  Huber,  Pavy, 
and  recently  also  Pick  and  Bial,^  proved,  by  a  diastiatic  enzyme  which, 
according  to  Rohmann  and  Borchardt,^  is  identical  with  a  diastatic 
enzyme  of  the  blood. 

This  post-mortem  sugar  formation  led  Bernard  to  the  assumption  of 
the  formation  of  sugar  from  glycogen  in  the  liver  during  life.  Bernard 
suggested  the  following  arguments  for  this  theory :  The  liver  always  con- 
tains some  sugar  under  physiological  conditions,  and  the  blood  from  the 
hepatic  vein  is  always  somewhat  richer  in  sugar  than  the  blood  from  the 
portal  vein.  The  correctness  of  either  or  both  of  these  statements  has 
been  disputed  by  many  investigators.  Pavy,  Ritter,  Schiff,  Eulen- 
BERG,  Lussana,  Abeles,  and  others  deny  the  occurrence  of  sugar  in  the 
liver  during  life,  and  the  greater  amount  of  dextrose  in  the  blood  from  the 
hepatic  vein  is  likewise  disputed  by  them  and  certain  other  investigators.^ 


'  See  Minkowski  and  Laves,  Arch.  f.  exp.  Patli.  ii.  Pliarm.,  23;  Kiilz,  Zeitsclir.  f. 
Biologic,  27. 

^  See  Dastre,  Compt.  rend,  de  Soc.  bid.,  47,  280,  and  Kaufmann,  ibid.,  316. 

^  Arthus  and  Huber,  Arch,  de  Physiol.  (5),  4,  659;  Pavy,  Journal  of  Physiol.,  22; 
Pick,  Hofmeister's  Beitr.,  3;   Bial,  Arch.  f.  (Anat.  u.)  Phy.siol.,  1901. 

^  Rohmann,  Verh.  d.  Ges.  deutsch.  Naturf.  u.  Arzte,  Breslau,  1903;  Borchardt, 
Pflfiger's  Arch.,  100. 

^  In  regard  to  the  literature  on  sugar  formation  in  the  liver  see  Bernard,  Legons  sur 
le  diab^te,  Paris,  1877;  Seegen,  Die  Zuckerbildtmg  ira  Tierkorper,  2.  Aufl.,  Berlin, 
1900;   M.  Bial,  Pfliiger's  Arch.,  55,  434. 


296  THE    LIVER. 

It  can  be  said  that  at  present  there  are  two  contradictory  views  on  the 
destruction  of  the  glycogen  in  the  Hving  organism:  Pavy's  view,  that  the 
glycogen  is  directly  used  without  being  previously  transformed  into  sugar, 
and  Bernard's  view,  which  is  accepted  by  most  investigators,  that  the 
glycogen  is  first  transformed  into  sugar  by  the  aid  of  diastatic  enzymes. 
According  to  certain  experimenters  (Dastre,  Noel-Paton,  E.  Cavazzani  ^), 
who  also  admit  a  destruction  of  the  glycogen  with  the  formation  of  sugar, 
the  change  is  not  brought  about  by  an  enzyme,  but  by  a  special  protoplasmic 
activity. 

The  doctrine  as  to  the  physiological  formation  of  sugar  in  the  liver  has 
obtained  an  energetic  advocate  in  Seegen.  He  maintains,  after  numerous 
experiments,  that  the  liver  regularly  contains  considerable  amounts  of 
sugar.  He  has  observed  an  increase  of  3  per  cent  in  the  quantity  of  dex- 
trose in  the  liver  of  a  dog  kept  alive  by  passing  arterial  blood  through  the 
organ,  and  lastly  he  has  also  found  in  a  very  great  number  of  experiments 
on  dogs  that  the  blood  from  the  hepatic  vein  always  contains  more — even 
double  as  much— sugar  than  the  blood  from  the  portal  vein.  Mosse  and 
ZuNTZ^  have  recently  made  objections  as  to  the  correctness  of  this  last 
statement,  and  it  follows  from  the  various  researches  on  this  question  that 
when  disturbing  influences  are  prevented,  the  blood  from  the  hepatic  vein 
is  only  very  little  richer  in  sugar  than  the  blood  from  the  portal  vein. 

Although  Seegen  energetically  espouses  the  doctrine  of  Bernard  as  to 
the  vital  sugar  formation  in  the  liver,  still  he  deviates  essentially  from 
Bernard  in  that  he  claims  the  sugar  is  not  derived  from  the  glycogen. 
According  to  Seegen,  the  sugar  is  formed  from  protein  and  fat.  His  older 
idea,  that  this  protein  was  peptone,  he  has  discarded.  Of  importance  for 
the  study  of  the  sugar  formation  in  the  liver  is,  on  the  contrary,  the  fact 
that  Seegen  has  found  a  substance  in  the  liver,  besides  glycogen,  which 
yields  dextrose  on  heating  with  dilute  acids.  He,  in  connection  with 
Neimann,  has  isolated  this  substance  in  the  form  of  a  nitrogenous  carbo- 
hydrate. O.  Simon  ^  has  also  recently  isolated  from  the  liver  a  proteose- 
like  substance  which  reduces  directly  and  yields  a  fermentable  sugar  on 
boiling  with  acids,  and  this  sugar  gives  an  osazone  melting  at  190°. 

Seegen  claims  to  have  shown  a  formation  of  sugar  from  fat  by  a  direct 
experiment  with  surviving  liver  tissue.  Certain  investigations  of  Weiss 
seem  to  substantiate  this  view,  while  other  experiments  of  Montuori,  Abder- 
halden,  and  Rona  and  Hesse  contradict  this  assumption.     Hildesheim  and 

*  In  regard  to  the  literature  see  Pick,  Hofmeister's  Beitrage,  3. 

*  Seegen,  Die  Zuckerbildung,  etc.,  and  Centralbl.  f.  Physiol.,  10,  497  and  822; 
Zuntz,  ibid.,  561;    Mosse,  Pfluger's  Arch.,  03;    Bing,  Skand.  Arch.  f.  Physiol.,  9. 

^  Seegen,  Arch.  f.  (Anat.  u.)  Physiol.,  1903;  Seegen  and  Neimann,  Wien.  Sitzungs- 
ber.,  112  (1903);  Simon,  Arch.  f.  exp.  Path.  u.  Pharm.,  49.  (See  glucothionic  acid, 
page  285.) 


GLYCOSURIAS.  297 

Leathes^  have  made  experiments  with  hver  pulp  which  indicate  tiie 
reverse,  i.e.,  the  formation  of  fat  from  gl^'cogen. 

The  circumstance  that  the  blood-sugar  rapidly  sinks  to  |-^  of  its  orig- 
inal quantity,  or  even  disappears  when  the  Uver  is  cut  out  of  the  circulation, 
speaks  for  a  \dtal  formation  of  sugar  in  the  hver  (Seegex,  Bock  and 
HoFFMAXx;  K\UF.\L\xx;  Taxgl  and  Harley;  Pavy).  In  geese  whose 
hvers  were  removed  from  the  circulation,  AIixkowski  found  no  sugar  in 
the  blood  after  a  few  hours.  On  removing  the  liver  from  the  circulation 
by  tying  all  the  vessels  to  and  from  the  organ,  the  quantity  of  sugar  in  the 
blood  on  drawing  is  not  increased  (Schexck^).  We  shall  also  learn  shortly 
of  certain  poisons  and  operative  changes  which  ma}'  cause  an  abundant 
ehmination  of  sugar,  but  only  when  the  liver  contains  gl3^cogen.  If  we  re- 
call the  fact  shown  by  Rohmann  and  Bial  ^  that  the  lymph  as  well  as  the 
blood  contains  a  diastatic  enz^^me,  then  several  reasons  speak  for'  the  view 
of  Berxard  that  the  post-mortem  formation  of  sugar  from  the  glycogen 
in  the  liver  is  a  continuation  of  the  vital  process. 

The  relationship  of  the  sugar  eliminated  in  the  urine  under  certain 
conditions,  such  as  in  diabetes  melUtus,  certain  intoxications,  lesions  of 
the  nervous  system,  etc.,  to  the  glycogen  of  the  liver  is  also  an  important 
question. 

It  does  not  enter  into  the  plan  and  scope  of  this  book  to  discuss  in 
detail  the  various  x-iews  in  regard  to  glycosuria  and  diabetes.  The  appear- 
ance of  dextrose  in  the  urine  is  a  S3'mptom  which  may  have  essentially 
different  causes,  depending  upon  different  circumstances.  Only  a  few  of 
the  most  important  points  will  be  mentioned. 

The  blood  contains  always  about  an  average  of  1.5  p.  m.,  while  the 
urine  has  in  it  at  most  only  traces  of  dextrose.  When  the  quantity  of  sugar 
in  the  blood  rises  to  .3  p.  m.  or  above,  then  sugar  passes  into  the  urine.  The 
kidneys  have  the  property  to  a  certain  extent  of  preventing  the  passage  of 
blood-sugar  into  the  urine;  and  it  follows  from  this  that  an  elimination 
of  sugar  in  the  urine  may  be  caused  partly  b}-  a  reduction  or  suppression 
of  this  above-mentioned  activity,  and  partly  also  by  an  abnormal  increase 
of  the  quantity  of  sugar  in  the  blood. 

The  first  seems,  according  to  v.  AIerixg  and  Minkow^ski,  to  be  the 
case  in  phlorhizin  diabetes,  v.  ]Merixg  has  found  that  a  strong  glycosuria 
appears  in  man  and  animals  on  the  administration  of  the  glucoside  phlor- 

*  Weiss,  Zeitschr.  f.  physiol.  Chem.,  24;  Montuori,  Maly's  Jahresb.,  26;  Abder- 
halden  and  Rona,  Zeitschr.  f.  physiol.  Chem.,  41;  Hesse,  Zeitschr.  f.  exp.  Path.  u. 
Therap.,  1;   Hildesheim  and  Leathes,  Journ.  of  Physiol.,  31. 

^  Seegen,  Bock,  and  Hoffmann,  see  Seegen,  1.  c;  Kaufmann,  Arch,  de  Physiol.  (5), 
8;  Tangl  and  Harley,  Pfliiger's  Arch.,  61;  Pavy,  Journ.  of  Physiol.,  29;  Minkowski, 
Arch.  f.  exp.  Path.  u.  Pharm.,  21;  Schenck,  Pfliiger's  Arch.,  57. 

'  See  foot-note  4,  p.  295. 


298  THE  LIVER, 

hizin.  The  sugar  eliminated  is  not  derived  from  the  gkicoside  alone.  It 
is  formed  in  the  animal  body,  and  in  fact,  at  least  on  prolonged  starvation, 
from  the  protein  substances  of  the  body.  The  quantity  of  sugar  in  the 
blood  is  not  increased,  but  rather  diminished,  in  phlorhizin  diabetes  (Min- 
kowski), but  this  is  disputed  by  Pavy.  Tliis  last  investigator  found, 
although  only  to  a  slight  degree,  that  the  sugar  in  the  blood  was  increased, 
but  he  holds  the  same  \new  that  v.  Mering  does,  that  phlorhizin  diabetes 
is  a  kidney  diabetes.  That  after  extirpation  of  the  kidney  in  phlorhizin 
diabetics  no  rise  in  the  blood-sugar  is  observed,  and  that  after  the  injection 
of  phlorhizin  in  the  renal  arteiy  of  one  side  the  urine  secreted  by  this  kidney 
contains  sugar  sooner  and  more  abundantly  than  the  urine  from  the  other 
kidney  (Zuntz),  speaks  in  favor  of  this  view.  The  experiments  especially 
performed  by  Pavy,  Brodie,  and  Siau  ^  upon  blood  containing  phlorhizin 
and  surviving  kidneys  also  indicate  the  same,  namely,  that  the  phlorhizin  acts 
upon  the  kidneys.  While  v.  jMering  believes  in  an  increased  permeabihty 
of  the  kidneys  for  sugar,  produced  by  the  phlorhizin,  Pavy  is,  on  the  con- 
trary', of  the  opinion  that  the  kidneys,  under  the  influence  of  the  phlorhizin, 
split  off  sugar  from  a  substance  circulating  in  the  blood,  perhaps  from  a 
proteid  with  loosely  combined  carbohydrate  groups. 

With  the  exception  of  phlorhizin  diabetes,  which  is  dependent,  accord- 
ing to  the  ordinaiy  views,  upon  a  change  or  special  processes  in  the  kidneys, 
and  in  which  no  essential  rise  in  the  blood-sugar  occurs,  all  other  forms  of 
glycosuria  or  diabetes,  as  far  as  known  at  present,  depend  on  a  hypergluccemia. 

A  hyperglucsemia  may  be  caused  in  various  ways.  It  may  be  caused, 
for  example,  by  the  introduction  of  more  sugar  than  the  body  can  destroy. 

The  ability  of  the  animal  body  to  assimilate  the  different  varieties 
of  sugar  has  naturally  a  hmit.  If  too  much  sugar  is  introduced  into  the 
intestinal  tract  at  one  time,  so  that  the  so-called  assimilation  Hmit  (see 
Chapter  IX,  on  absorption)  is  overreached,  then  the  excess  of  absorbed 
sugar  passes  into  the  urine.  This  form  of  glycosuria  is  called  alimentary 
glycosuria,^  and  it  is  caused  by  the  passage  of  more  sugar  into  the  blood 
than  the  liver  and  other  organs  can  destroy. 

*  In  regard  to  the  literature  on  phlorhizin  diabetes  see  v.  Mering,  Zeitschr.  f.  klin. 
Med.,  14  and  16;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Moritz  and  Prausnitz, 
Zeitschr.  f.  Biologie,  27  and  29;  Kiilz  and  Wright,  ibid.,  27,  181;  Cremer  and  Ritter, 
ibid.,  28  and  29;  Contejean,  Compt.  rend,  de  soc.  biol.,  48;  Lusk,  Zeitschr.  f.  Biologie, 
36;  Levene,  Journal  of  Physiol.,  17;  Pavy,  ibid.,  20,  and  with  Brodie  and  Siau,  29; 
Arteaga,  Amer.  Journ.  of  Physiol.,  6;  O.  Loewi,  Arch.  f.  exp.  Path.  u.  Pharm.,  47; 
N.  Zuntz,  Arch.  f.  (Anat.  u.)  Physiok,  1895;  Stiles  and  Lusk,  Amer.  Journ.  of  Physiol., 
10;   Cremer,  Ergebnisse  der  Physiol.,  1,  Abt.  1,  and  the  monographs  upon  diabetes. 

^  In  regard  to  alimentary  glycosuria  see  Moritz,  Arch.  f.  klin.  Med.,  46,  which  also 
contains  the  older  literature;  B.  Rosenberg,  Ueber  das  Vorkommen  der  alimentaren 
Glykosurie,  etc.  (Inaug.-Dissert.  Berlin,  1897);  van  Oondt,  Miinch.  med.  Wochen- 
schr.,  1898;   v.  Noorden,  Die  Zuckerkrankheit,  3.  Aufl.,  1901. 


GLYCOSURIAS.  299 

As  the  liver  cannot  transform  into  glycogen  all  the  sugar  which  comes 
to  it  in  ahmentan,-  glycosuria,  it  is  possible  that  a  glycosuria  may  be  pro- 
duced also  under  pathological  conditions,  even  by  a  moderate  amount  of 
carbohydrate  (100  grams  dextrose),  which  a  healthy  person  could  overcome. 
Tliis  is  the  case,  among  others,  in  various  affections  of  the  cerebral  system 
and  in  certain  chronic  poisonings.  Certain  observers  include  the  lighter 
forms  of  diabetes  in  this  class  of  glycosiiria. 

We  differentiate  between  Ught  and  severe  forms  of  diabetes.  In  the 
first  the  urine  contains  sugar  only  when  carbohydrates  are  taken  as  food, 
wliile  in  the  other  case  the  urine  contains  sugar  even  -uith  food  entirely 
free  from  carbohydrates.  According  to  the  views  of  several  in^•estigators, 
in  light  forms  of  diabetes  the  hver  is  incapable  of  transforming  into 
gh  cogen  all  the  carbohydrates  introduced,  or  to  utilize  this  glycogen  in  a 
normal  way,  and  the  acti\ity  of  the  Hver-cells  is  also  reduced  or  changed  in 
these  cases. 

A  hyperglucsemia  which  passes  into  a  glycosuria  may  also  be  brought 
about  by  an  excessive  formation  of  sugar  from  the  glycogen  and  other 
substances  within  the  animal  body. 

The  so-called  piqilre,  and  also  probably  those  glycosurias  which  occur 
after  other  lesions  of  the  ner^'ous  system,  belong  to  the  above  group  of 
glycosurias.  The  glycosuria  produced  on  poisoning  Anth  carbon  monox- 
ide, adrenahn,  curare,  str}-chnine,  morphine,  etc.,  also  belongs  to  this  group. 
That  the  glycosuria  produced  in  certain  cases,  as  after  piqilre.  is  due 
to  an  increased  transformation  of  the  glycogen  follows  from  the  fact  that 
no  glycosuria  appears,  under  the  above-mentioned  circumstances,  when 
the  liver  has  been  pre\'ioush-  made  free  from  glycogen  by  star^'ation  or 
other  means.  In  other  cases,  as  in  carbon-monoxide  poisoning,  the  sugar 
is  probably  derived  from  the  proteins,  ecause  glycosuria  boccurs  only  in 
those  cases  where  the  poisoned  animal  has  a  sufficient  quantity  of  protein 
at  its  disposal  (Straub  and  Rosexsteix^).  Protein  starvation  \\ith  a 
simultaneously  abundant  supply  of  carbohydrates  causes  this  glycosuria  to 
disappear. 

A  hyperglucsemia  \dth.  glycosuria  may  also  be  caused  by  a  decreased 
ability  of  the  animal  body  to  consume  or  destroy  the  sugar.  In  this  case 
the  sugar  must  accumulate  in  the  blcod.  and  the  formation  of  severe  cases 
of  diabetes  meUitus  is  now  generally  explained  by  this  process. 

The  inability  of  diabetics  to  destro}-  or  consume  the  sugar  does  not 
seem  to  be  connected  "uith  anj-  decrease  in  the  oxidative  energy-  of  the 
cells.     The  oxidative  processes  are    not  diminished  generally  in  diabetics 

'  See  Dock,  Pfliiger's  Arch.,  5;  Bock  and  Hoffmann,  Exp.  Studien  iiber  Diabetes 
(Berlin,  1874);  CI.  Bernard,  Le§ons  snr  le  diabete  (Paris);  T.  Araki,  Zeitschr.  f.  physiol. 
Chem.,  15,  351;  Straub,  Arch.  f.  exp.  Path.  u.  Pharm.,  3S;  Rosenstein,  ibid..  40; 
Pfiiiger,  Pfliiger's  Arch.,  96. 


300  THE   LIVER. 

(ScHULTZEN,  Nencki  and  Sieber^),  and  this  has  recently  been  substan- 
tiated b}'  Baumgarten.  This  latter  investigator  made  experiments  ^\ith 
several  bodies  which  on  account  of  their  aldehyde  nature  were  closely 
related  to  sugar  or  were  cleavage  or  oxidation  products  of  the  same, 
namely,  glucuronic  acid,  d-gluconic  acid,  c^-saccharic  acid,  glucosamine, 
mucic  acid,  and  others,  and  he  found  that  diabetics  destroyed  or  burnt  these 
bodies  to  the  same  extent  as  healthy  individuals.  Besides  tliis  it  must 
be  remarked  that  the  two  varieties  of  sugar,  dextrose  and  le\'ulose,  which 
are  oxidized  with  the  same  readiness,  act  differently  in  diabetics.  Accord- 
ing to  KuLZ  and  other  investigators  levulose  is,  contrary  to  dextrose, 
utilized  to  a  great  extent  in  the  organism,  and  may,  according  to  ^Iinkowski,^ 
even  cause  a  deposit  of  glycogen  in  the  liver  in  animals  with  pancreas 
diabetes  (see  telow).  The  combustion  of  protein  and  fat  takes  place  as 
in  healthy  subjects,  and  the  fat  is  completely  burned  into  carbon  dioxide 
and  water.  In  this  diabetes  the  ability  of  the  cells  to  utilize  especially 
the  dextrose  suffers  diminution,  and  the  explanation  of  this  has  teen 
sought  in  the  fact  that  the  dextrose  is  not  previously  split  before  com- 
bustion. 

CO 

The  variation  in  the  respiratory  quotient,  i.e.,  the  relation  — ^,  seems 

to  show  an  insufficiency  of  the  dextrose  combustion  in  the  tissues  in  diabetes. 
As  "v^dll  be  thoroughly  explained  in  a  following  chapter,  this  quotient  is 
greater  the  more  carbohydrates  are  burnt  in  the  body,  and  it  is  correspond- 
ingly smaller  when  protein  and  fat  are  chiefly  burnt.  The  investigations 
of  Leo,  Hanriot,  Weintraud  and  Laves,^  and  others  have  shown  that 
in  severe  cases  of  diabetes,  in  the  starving  condition  the  low  quotient  is 
not  raised  after  partaking  of  dextrose,  as  in  healthy  individuals,  but  that 
it  is  raised  after  feeding  le\ailose,  which  is  also  of  value  to  diabetics  (Wein- 
traud and  Laves).  The  povertj^  of  the  organs  and  tissues  of  diabetics 
in  glycogen  shows  that  not  only  is  the  combustion  of  the  dextrose  dimin- 
ished, but  also  the  transformation  of  the  same  into  glycogen,  and  its  valu- 
ation as  a  whole  is  decreased. 

There  are  also  certain  investigators  who  consider  that  diabetes  is  due 
to  an  increased  production  of  sugar  in  the  liver — a  view  which  has  received 
some  support  in  the  artificially  produced  pancreatic  diabetes. 

The  investigations  of  ^Minkowski,  v.  Mering,  Domenicis,  and  later 

'  Schultzen,  Berl.  klin.  Wochenschr.,  1872;  Nencki  and  Sieber,  Journ.  f.  prakt. 
Chem.  (X.  F.),  26,  35;  Baumgarten,  "Ein  Beitrag  zur  Kenntniss  des  Diabetes  mel- 
litus,"  Habilitationsschrift,  also  Zeitschr.  f.  exp.  Path.  u.  Therap.,  2,  1905. 

^  Kiilz,  Beitrage  zur  Path.  u.  Therap.  des  Diabetes  melHtus  (Marburg,  1874),  1; 
Weintraud  and  Laves,  Zeitschr.  f.  physiol.  Chem.,  19;  Haycraft,  ibid.;  Minkowski, 
Arch.  f.  exp.  Path.  u.  Pharm.,  31. 

3  See  V.  Noorden,  Die  Zuckerkrankheit,  .3.  Aufl.,  1901. 


PAI\CRE.\S  AND  GLYCOLYSIS.  301 

of  man}'  other  investigators  ^  have  shown  that  a  true  diabetes  of  a  severe 
kind  is  caused  b}'  the  total  or  nearly  total  extirpation  of  the  pancreas  of 
many  animals,  especiall}''  dogs.  As  in  man  in  severe  forms  of  diabetes, 
so  also  in  dogs  with  pancreatic  diabetes,  an  almndant  elimination  of  sugar 
talces  place  even  on  the  complete  exclusion  of  carbohydrates  from  the 
food. 

Artificial  pancreas  diabetes  may,  at  least  in  cases  where  the  pancreas 
has  not  been  completely  extirpated,  present  exactly  the  same  conditions 
as  diabetes  in  man,  but  opinions  differ  as  to  the  cause  of  this  diabetes. 
It  is  generally  accepted  that  in  pancreas  diabetes  a  diminished  consumption 
of  sugar  takes  place;  but  there  are  several  investigators  who  are  of  another 
opinion  and  who  explain  this  form  of  diabetes  as  due  at  least  not  entirely 
to  a  diminished  combustion  of  sugar,  but  to  a  diseased  increase  in  the 
sugar  formation.  From  tliis  it  follows  that  the  pancreatic  gland  exerts  on 
the  formation  of  sugar  in  the  liver  a  regulating  action  which  is  absent  on 
the  extirpation  of  the  gland. 

]\Iany  important  observations  show  that  a  close  relation  exists  between 
the  liver  and  pancreas  diabetes.  Pfluger  has  also  shown  that  especially 
in  diabetes  produced  by  Saxdmeyer's  method  (partial  extirpation  with 
subsequent  destruction  of  the  remains  of  the  gland  in  the  abdominal  cavity, 
when  the  animal  remains  alive  for  a  longer  time  than  after  total  extir- 
pation) the  liver  does  not  lose  weight,  although  the  total  weight  of  the 
animal  diminishes  greatly,  wliile  in  starvation  ^\'ithout  diabetes  the  hver 
loses  weight  more  than  the  other  parts  of  the  body.  PFLtJGER  concludes 
from  this  that  the  hver  in  diabetes  works  actively  and  is  the  most 
important  seat  of  production  of  diabetic  sugar. 

We  do  not  know  how  the  pancreas  acts  in  the  formation  or  the  de- 
struction of  sugar,  and  we  have  essentially  two  contradictoiy  \-iews  on 
tliis  subject.  According  to  one  view  the  action  is  of  a  nervous  kind,  while 
the  other  ^•iew  is  that  we  are  deaUng  \sith  an  internal  secretion  of  special 
bodies  wliich  in  an  unknown  manner  perhaps  act  upon  the  nerve  centres 
and  regulate  the  formation  or  the  destruction  of  sugar.  The  assumption 
of  an  internal  secretion  is  rather  generally  accepted  and  is  based  on 
the  investigations  of  Minkowski.  Hedox,  Laxceraux.  Thiroloix.  and 
others  2  upon  the  action  of  the  subcutaneous  transplantation  of  the  gland. 
According  to  these  investigations  a  subcutaneously  transplanted  piece  of 

'  See  Minkowski,  Untersuchungen  iiber  Diabetes  mellitus  nach  Exstirpation  des 
Pankreas  (Leipzig,  1S93);  v.  Xoorden,  Die  Zuckerkrankheit  (Berlin,  1901),  which 
contains  a  very  copious  index  of  the  literature.  In  regard  to  diabetes  see  also  CI. 
Bernard,  Legons  sur  le  diabete  (Paris);  "Seegen,  Die  Zuckerbildung  ini  Thierkorper 
(Berlin,  1890),  and  Pfliiger,  Das  Glykogen,  2.  Aufl.,  1905. 

'  See  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Hedon,  Diabete  pancreatique, 
Travaux  de  Physiologie  (Laboratoire  de  Montpellier,  1898),  and  the  works  on  diabetes. 


302  THE   LIVER. 

the  gland  can  completely  perform  the  functions  of  the  pancreas  as  to  the 
sugar  exchange  and  the  sugar  elimination,  because  on  the  removal  of  the 
intra-abdominal  piece  of  gland  the  animal  in  this  case  does  not  become 
diabetic.  But  if  the  subcutaneously  embedded  piece  of  pancreas  is  then 
subsequently  removed,  an  active  elimination  of  sugar  appears  immediately. 
PFLtJGER  has  made  important  objections  to  the  force  of  proof  in  these 
experiments. 

This  internal  secretion  of  the  pancreas  has  in  recent  times  been  sup- 
posed to  be  connected  with  the  so-called  islands  of  Langerhans;  but  no 
positive  results  have  been  obtained  in  this  connection.^  We  are  also  not 
acquainted  mth  the  kind  of  active  substance  here  formed. 

The  glycolytic  property  of  the  blood  as  shown  by  Lupine  was  con- 
si(iered  for  a  time  to  be  due  to  a  glycolytic  enzyme  formed  in  the  pancreas, 
and  pancreas  diabetes  used  to  be  explained  by  the  fact  that  the  action 
of  this  enzyme  was  removed  when  the  gland  was  extirpated.  This  gly- 
colysis is  not  sufficient,  even  if  it  is  derived  from  the  pancreas,  to  explain 
the  transformation  of  the  large  quantity  of  sugar  in  the  body,  and  for  the 
destenction  of  sugar  we  are  also  obliged  to  accept  a  glycolysis  in  the  organs 
and  tissues.  The  views  in  regard  to  this  glycolysis  differ  in  certain  points. 
According  to  one  view  (Spitzer  and  others)  special  oxidases  are  active 
in  the  glycolysis,  while  another  view  (Stoklasa)  considers  the  glycolysis 
as  analogous  to  alcoholic  fermentation,  where  we  have  processes  brought 
on  by  special  tissue  zymases  (see  Chapter  I). 

Another  important  question  is  whether  one  organ  can  bring  about 
glycolysis  or  wheiher  a  combination  of  organs  is  required.  Cohnheim 
has  found  that  a  cell  free  fluid  can  be  obtained  from  a  mixture  of  pan- 
creas and  muscle,  which  destroys  dextrose,  while  the  pancreas  alone  does 
not  have  this  action  and  the  muscle  only  to  a  slight  extent.  The  pan- 
creas does  not  contain,  according  to  Cohnheim,  a  glycolytic  enzyme,  but 
a  substance  resistant  to  boiling  temperatures,  which  is  soluble  in  water  and 
alcohol,  and  which,  like  an  amboceptor,  activates  a  glycolytic  proenzyme 
which  exists  in  the  muscle  fluid,  but  which  is  inactive  alone  and  which 
retards  glycolysis  when  it  exists  in  excess.  De  Meyer  holds  a  nearly 
similar  view,  but  with  this  exception,  that  he  does  not  consider  that  the 
activating  substance  comes  from  the  muscles  but  from  the  leucocytes. 
LUPINE  2  has  also  expressed  the  opinion  that  the  pancreas  does  not  have 
a  direct  glycolytic  action  by  internal  secretion,  but  more  likely  by  the 
glycolysis  encouraged  by  the  action  of  cell  protoplasm. 

'  See  Diamare  and  Kubiabko,  Centralbl.  f.  Physiol.,  18,  and  Diamare,  ibid.,  19, 
Rennie,  ibid.,  18;  Sauerbeck,  Vnchow's  Arch  ,  177. 

'  Cchnheirrj,  Zeitbchr.  f.  phjbiol.  Chera.,  39,  42,  4.3,  and  47;  Do  Meyer,  Arch  iniern. 
de  Physiol.,  2,  cited  from  Bicchem   Centralbl.,  3. 


SUGAR   FORMATION    FROM    PROTEIN.  303 

The  statements  of  Cohnheim  have  not  been  fully  confirmed  by  other 
investigators.  On  the  contrar}-,  several  investigators,  Stoklasa  and 
collaborators,  Feixschmidt,  Aexheim  and  Rosexbaum,  and  Brauxstein,^ 
could  not  detect  any  glycolytic  activity  either  in  the  pancreas  alone  or 
in  muscles  and  other  organs  alone  (with  the  exclusion  of  bacteria).  The 
liver  also  belongs  to  these  organs,  in  which,  it  must  be  remarked,  the  gly- 
colytically  active  substance  has  been  absent  in  severe  cases  of  diabetes. 
CoHXHEiAi's  statements  have,  on  the  contrary-,  been  substantiated  in  part 
by  Arnheii.i  and  Rosexbaum  and  R.  Hirsch,  who  find  that  the  pancreas 
has  the  power  of  raising  the  glycolytic  action  of  the  liver  and  the  muscles. 
On  the  other  hand,  Glaus  and  Embdex  have  not  been  able  to  obtain  the 
activating  action  of  the  pancreas  upon  muscle-juice,  but  according  to 
CoHXHEiM  this  is  probably  due  to  the  fact  that  these  investigators  added 
too  large  quantities  of  pancreas,  whereby  the  retarding  action  came  into 
effect.  No  positive  conclusions  on  the  mode  of  action  of  the  pancreas 
in  sugar  destruction  or  sugar  formation  can  be  drawn  from  these  con- 
tradictory statements. 

Where  does  the  sugar  eliminated  in  diabetes  originate?  Does  it  depend 
entirely  upon  the  carbohydrates  of  the  food  or  the  store  of  carbohydrate 
in  the  body,  or  has  the  body  the  power  of  producing  sugar  from  other 
material?  To  Luthje  belongs  the  credit  for  positively  deciding  this 
question.  He  has  made  experiments  on  dogs  with  pancreas  diabetes,  in 
which  on  a  protein  diet  free  from  carbohydrates  so  much  sugar  was  elim- 
inated that  it  could  not  possibly  be  accounted  for  by  the  store  of  glycogen 
or  other  carbohydrate-containing  substances  in  the  body.  Similar  experi- 
ments have  also  been  performed  later  by  Pfluger,^  and  the  power  of  the 
animal  body  to  produce  sugar  from  non-carbohydrate  material  is  now 
definitely  proven. 

Is  this  sugar  produced  from  protein  or  fat,  or  from  both?  This  ques- 
tion so  far  has  not  been  answered,  and  it  is  the  subject  of  continuous  dis- 
pute. It  is  not  possible  to  enter  into  an  exhaustive  and  detailed  discus- 
sion of  the  question  in  a  text-book,  and  we  will  only  mention  briefly 
certain  of  the  most  important  observations  and  historical  points. 

The  largest  amount  of  sugar  which  we  can  obtain  theoretically  from 
protein  is  8  grams  of  sugar  from  1  gram  of  protein  nitrogen  if  we  admit  that 
all  the  carbon  of  the  protein,  with  the  exception  of  that  necessan,-  to  form 
ammonium  carbonate,  is  used  for  the  formation  of  sugar.     The  actual  rela- 

'  Stoklasa  and  collaborators,  Centralbl.  f.  Physiol.,  17,  and  Ber.  d.  d.  ehem.  Gesellsch., 
36  and  38;  Feinschmidt,  Hofmeister'e  Beitrage,  4;  Hirsch,  ibid.;  Claus  and  Embden, 
ibid.,  6;  Arnheim  and  Rosenbaum,  Zeitschr.  f.  physiol.  Cbem.,  40;  Braunstein, 
Zeitschr   f.  klin.  Med..  51. 

'  Liitbje,  Deuls-cb  Arch  f.  klin.  Med  ,  79,  and  Pfliiger's  Arch.,  106;  Pfliiger, 
Pfliiger.-  Arrb     lOS 


304  THE   LIVER. 

tion  between  dextrose  and  nitrogen  in  the  urine,  i.e.,  the  quotient  D:N,  has 
been  repeatedly  determined  in  various  forms  of  diabetes.  In  a  large  number 
of  cases  this  has  been  found  to  be  equal  to  2.8  to  3.8.  It  may  undergo 
considerable  variation,  and  in  certain  cases  it  may  indeed  be  loAver  than  1 
as  well  as  higher  than  8.  From  these  quotients  conclusions  have  been 
drawn  as  to  the  amount  of  sugar  formed,  as  well  as  the  origin  of  the  sugar, 
but  according  to  the  \dews  of  Hammarsten  such  conclusions  are  mostly 
very  uncertain.  The  sugar  eliminated  by  the  urine  represents  the  differ- 
ence between  the  total  sugar  production  of  the  body  and  the  quantity 
of  sugar  burned  or  utilized.  Only  under  the  su])position  that  the  body 
cannot  burn  or  utilize  any  sugar  is  the  sugar  of  the  urine  a  measure  of 
the  quantity  of  sugar  produced;  it  is  not  known  how  far  this  supposition 
can  be  applied  in  the  various  forms  of  diabetes.  Still  several  observa- 
tions seem  to  show  that  in  the  different  forms  of  diabetes  variable  amounts 
of  the  sugar  are  burned.  A  sugar  formation  from  fat  can  be  presumed  only 
when  the  quotient  is  specially  high. 

The  property  of  protein  of  increasing  the  elimination  of  sugar  is  con- 
sidered as  an  important  proof  of  the  formation  of  sugar  from  protein.  In 
this  regard  those  experiments  are  of  special  interest  in  which  the  diabetic 
animal  is  allowed  to  starve  until  the  urine  is  poor  in  sugar  or  indeed  free 
from  sugar,  and  then  by  feeding  with  protein  an  abundant  elimination  of 
sugar  is  produced.  If  we  do  not  want  to  accept  in  this  case  that  the  pro- 
tein, but  rather  the  fat,  was  the  material  from  which  the  sugar  was  pro- 
duced, still  we  must  admit  eitlier  of  a  sugar-sparing  action  due  to  protein 
or  of  a  strong  sugar  formation  from  fat,  incited  by  the  protein. 

A  sparing  in  the  sense  that  the  protein  is  oxidized  instead  of  the  sugar, 
and  in  this  manner  protects  it,  is  naturally  possible  only  under  the  sup- 
position that  the  body  can  burn  at  least  a  part  of  the  sugar,  otherwise 
there  would  be  nothing  to  spare  and  nothing  to  protect  from  burning. 
The  assumption  of  such  an  indirect  action  of  proteins  is  difficult  to  recon- 
cile with  the  common  view  of  the  inability  of  the  body  to  burn  sugar  in 
diabetes.  Luthje  ^  has  communicated  one  experiment  among  others,  in 
which  a  dog  with  pancreas  diabetes,  whose  weight  before  starvation  was 
18  kilos,  with  nineteen  days'  starvation  eUminated  an  average  of  10.4  grams 
sugar  for  the  last  six  days  of  starvation.  By  exclusive  protein  feeding 
the  quantity  of  sugar  per  day  could  be  raised  to  a  maximum  of  123.8  grams, 
and  as  average  it  was  97.5  grams  for  the  ten  protein  days.  The  protein 
therefore  had  protected  daily  an  average  of  87  grams  sugar  from  burning, 
wliich  is  hardly  possible;  and  if  in  the  diabetic  animal  we  admit  of  this 
considerable  power  of  burning  sugar  the  quotient  D:N  becomes  valueless 
as  a  measure  of  the  quantity  of  sugar  formed. 

'  Deutsch.  Arch.  f.  klin.  Med.,  79. 


SUGAR    FORMATION   FROM   FAT.  30o 

If.  on  the  contrary,  we  admit  of  an  indirect  action  of  proteins  in  that 
they  incite  a  sugar  formation  from  fat,  perhaps  by  a  certain  ven-  impor- 
tant increase  in  the  acti\ity  of  the  liver,  we  are  opposed  by  the  great 
difficulty  that,  according  to  known  laws  of  metabolism,  the  proteins  do 
not  raise  the  fat  metabolism,  but  rather  diminish  it.  The  protein  dis- 
places a  corresponding  quantity  of  fat  from  the  metabolism,  and  if  the 
fat  was  the  only  source  of  sugar  then  in  this  case  we  would  expect  a 
diminished  elimination  of  sugar  instead  of  an  increased  one.  Nevertheless 
the  above  action  of  protein  upon  sugar  ehmination  is  much  more  easily 
explained  b\"  the  assumption  of  a  sugar  formation  from  protein  than  from  fat. 

The  action  of  monamino-acids  upon  the  carlx)hydrate  metabohsm  has 
also  given  important  ground  for  the  assumption  of  a  sugar  formation  from 
protein.  That  a  deamidation  occtirs  in  the  animal  body  was  shown  by 
the  older  obsen.-ations  of  Bai:^l\xx  and  Blexdeemaxx.  Further  proofs  of 
this  were  furnished  b}-  the  recent  investigations  of  Xeubeeg  and  Laxg- 
STEES".  where  in  feeding  experiments  with  alanine  they  found  abundance  of 
lactic  acid  in  urine,  and  finally  Laxg  ^  has  shown  that  various  organs  in 
antiseptic  autolysis  have  the  power  of  deamidating  amides  and  amino-acids. 
As  from  amino-acids  by  deamidation  it  is  possible  to  produce  oxyfatty  acids 
according  to  the  formula  — CH.XH2-H20=— CHCOH^-f  XH3,  it  was  inter- 
esting to  test  the  action  of  amino-acids  upon  carbohydrate  metabolism. 
Several  investigations  have  been  carried  on  with  this  in  \'iew,  such  as  those 
of  L.\XGSTEix  and  Xeuberg,  R.  Cohx  and  F.  Kraus.  which  have  shown 
a  ver}-  probable  formation  of  carbohydrate  under  the  influence  of  amino- 
acids;  but  the  investigations  of  Embdex  and  Salomox  and  of  Embdex 
and  Alm-\gia2  have  positively  shown  in  a  dog  without  a  pancreas  that 
the  amino-acids  can  bring  about  a  re-formation  of  carbohydrate.  It  is 
still  an  open  question  whether  the  amino-acids  are  only  indirectly  active 
in  this  or  whether  they  form  the  material  from  which  the  sugar  is  formed. 
In  general  we  consider  the  formation  of  sugar  ^ith  amino-acids  as  inter- 
mediar\-  bodies  as  ver}-  probable. 

If  we  presume  a  formation  of  sugar  from  fat  we  must  differentiate 
between  the  two  components  of  neutral  fats,  that  is,  tetween  the  glycerine 
and  the  fatty  acids.  A  formation  of  sugar  from  glycerine  can  be  con- 
sidered as  proven  from  the  investigations  of  Ceemee.  and  especially  those 
of  Luthje.3  and  in  what  follows  we  will  discuss  only  the  formation  of 
sugar  from  the  fattv  acids. 

'  Baumann.  Zeitichr.  f.  physiol.  Chem..  4:  Blendermarm,  ibid..  G;  Xeuberg  and 
Langstein.  .\rch.  f.  (Anat.  u.)  Physiol.,  1903.  Suppl.;  Lang.  Hofmeister's  Beitrage,  5. 

^  Langstein  and  Xeuberg.  1.  c;  Cohn.  Zeitschr.  f.  physiol.  Chem..  2S:  F.  Ivraus, 
Berl.  klin.  Wochenschr..  1904;  Embden  and  Salomon,  Hofmeister's  Beitrage,  5  and 
6,  and  with^Almagia,  ibid..  7. 

'Cremer,  Sitzungsber.  d.  Ges.  f.  Morph.  u.  Physiol.  Munchen,  1902;  Luthje, 
Deutsch.  Arch.  f.  klin.  Med..  SO. 


306  THE   LIVER. 

The  formation  of  sugar  from  fat  seems  to  occur  in  the  plant  kingdom, 
and  as  the  chemical  processes  in  the  animal  and  plant  life  are  in  principle 
the  same  it  makes  the  possibility  of  a  sugar  formation  from  fat  very 
probable.  Such  an  origin  of  sugar  in  the  animal  body  is  accepted  by 
many  investigators,  especially  by  Pfluger  and  several  French  observers, 
among  whom  we  must  specially  mention  Chauveau  and  Kaufmann.^ 

Where  food  as  free  from  carbohydrate  as  possible  is  taken,  the  quo- 
tient D:  N  is  high,  i.e.,  higher  than  8,  as  well  as  when  the  quantity  of  sugar 
is  so  large  that  it  cannot  be  accounted  for  by  the  calculated  protein 
(and  carbohydrate)  metabohsm,  then  if  the  observations  are  otherwise  free 
from  error  we  can  admit  of  a  formation  of  sugar  from  fat.  Several  such 
cases  of  diabetes  in  man  have  been  published  (Rumpf,  Rosenqvist,  Mohr, 
v.  NooRDEN,  and  others),  and  also  in  animals  (Hartogh  and  Schumm).^ 
Although  these  researches  are  not  fully  conclusive,  still  certain  of  them 
indicate  a  probable  formation  of  sugar  from  fat.  We  also  have  several 
conditions  which  indicate  the  same,  namely,  that  in  phlorhizin  diabetes 
after  the  disappearance  of  the  liver-glycogen  the  fat  which  migrates  to  the 
liver  serves  as  material  for  the  formation  of  sugar  (PFLtJGER) ;  still  this  is 
not  sufficient  as  a  positive  proof. 

Starting  with  the  quotient  D:N,  which  he  sets  at  3.67,  Magxus-Levy 
has  calculated  the  quantity  of  oxygen  necessary  for  the  combustion  of  the 
protein,  provided  the  sugar  was  formed  therefrom,  and  also  the  quantity 
of  carbon  dioxide  j^roduced,  i.e.,  the  respiratory  quotient  for  these  cases. 
On  comparison  of  these  results  with  the  low  respiratory  quotient  observed 
in  diabetics,  he  comes  to  the  conclusion  that  the  sugar  is  derived  from  the 
protein.  Pfluger,^  who  has  made  a  different  calculation,  comes  to  an 
entirely  different  result,  and  considers  that  the  low  values  for  the  respira- 
tory quotient  in  diabetes  are  positive  proof  that  the  sugar  is  not  formed 
from  the  proteins,  but  from  the  fats.  As  the  quotient  D:N  is  not  an 
accurate  measure  of  the  quantity  of  sugar  formed,  and  as  we  cannot,  for 
the  present,  exactly  measure  the  quantity  of  oxygen  necessary  for  the 
formation  of  sugar  from  the  protein,  Hammarsten  believes  that  it  is  just 
as  impossible  to  conclude  from  the  respiratory  quotient  that  sugar  is 
formed  from  the  fats  as  from  the  proteids. 

We  have  no  exact  proofs  of  a  sugar  formation  from  fat  or  from  protein 
alone,  nevertheless  we  have  proofs  of  the  possibility  of  a  formation  from 
both  of  these.     There  is  really  no  objection  to  the  assumption  that  the  body 

'  Kaufmann,  Arch.  f.  Physiol.  (5),  S,  where  Chauveau's  work  is  cited. 

^  Rumpf,  Berl.  klin.  Wochenschr.,  1899;  Rosenqvist,  ibid.;  Mohr,  ibid.,  1901;  v. 
Noorden,  Die  Zuckerkrankheit,  3.  Aufl.,  Berlin,  1901;  Hartogh  and  Schumm,  Arch.  f. 
Path.  u.  Pharm.,  4.>.  See  also  the  works  of  O.  Loewi,  ibid.,  47,  and  Lusk,  Zeitschr.  f. 
Biologie,  42. 

^Magnus-Levy,  Zeitschr.  f.  klin.  Med.,  ')('»;    Pfliiger,  Pfluger's  Arch.,  108. 


BILE   AND   ITS  F0R:MATI0X.  307 

has  the  power  of  producmg  sugar  from  protein  as  well  as  from  fat.  The 
obsen-ations  on  the  formation  of  sugar  or  on  the  carbohydrate  metabolism 
in  diabetes  do  not  give  any  positive  explanations  as  to  the  question  whether 
proteins  are  direct  glycogen-formers  or  not. 


The  Bile  and  its  Formation. 

By  the  establishment  of  a  biliar}-  fistula,  an  operation  which  was  first 
performed  by  Schwann  in  1S44  and  which  has  been  improved  lately  by 
Dastre  and  Pawlow,i  it  is  possible  to  study  the  secretion  of  the  bile.  This 
secretion  is  continuous,  but  with  var}-ing  intensity.  It  takes  place  imder 
a  ver\'  low  pressure;  therefore  an  apparently  imimportant  hindrance  in  the 
outflow  of  the  bile,  namely,  a  stoppage  of  mucus  in  the  exit,  or  the  secretion 
of  large  quantities  of  viscous  bile,  may  cause  stagnation  and  absorption  of 
the  bile  by  means  of  the  lymphatic  vessels  (absorption  icterus). 

The  quantity  of  bile  secreted  in  the  twenty-four  hours  in  dogs  can  be 
exactly  determined.  The  quantity  secreted  by  different  animals  varies, 
and  the  limits  are  2.9-36.4  grams  of  bile  per  kilo  of  weight  in  the  twenty- 
four  hours.- 

The  statements  as  to  the  extent  of  bile  secretion  in  man  are  few  and 
not  to  be  depended  on.  Ranke  found  (using  a  method  which  is  not  free 
from  criticism)  a  secretion  of  14  grams  of  bile  with  0.44  gram  of  solids  per 
kilo  in  twenty-four  hours.  Xoel-Patox.  MayoRobsox,  H.imm-^esten, 
Pfaff  and  Balch.  and  Beaxd  ^  have  found  a  variation  between  514  and 
1083  c.c.  per  twenty-  f oiu:  hours.  Such  determinations  are  of  doubtful  value, 
because  in  most  cases  it  follows  from  the  composition  of  the  collected  bile 
that  the  fluid  is  not  the  result  of  a  secretion  of  normal  liver  bile. 

The  quantity  of  bile  secreted  is.  however,  as  specially  shown  by  St.u)EL- 
maxx,^  subject  to  such  great  variation  even  under  physiological  conditions 
that  the  study  of  those  circumstances  which  influence  the  secretion  is  ven- 
difficult  and  uncertain.  The  contradictory-  statements  by  different  investi- 
gators may  probably  be  explained  by  this  fact. 

In  stan-ation  the  secretion  diminishes.     According  to  LukJ-Ajxow  and 

'  Schwann.  Aich.  f.  (Anat.  u.)  Physiol.,  1S44;  Dastre.  .\rch.  de  Physiol.  (5),  2; 
Pawlow,  Ergebnisse  der  Physiol.,  1,  Abt.  1. 

-  In  regard  to  the  quantity  of  bUe  secreted  in  animals  see  Heidenhain,  Die  Gallenab- 
sonderung.  ia  Hermann's  Handbuch  der  Physiol.,  o,  and  Stadelmann,  Der  Icterus  xmd 
seine  verschiedenen  Formen  (Stuttgart,  1S91). 

'  Ranke.  Die  Blutvertheilimg  imd  der  Thatigkeitswechsel  der  Organe  (Leipzis. 
ISTl);  Xoel-Paton,  Rep.  Lab.  Roy.  Coll.  Edinburgh,  3;  Mayo-Robson,  Proc.  Roy.  Soc, 
47:  Hammarsten,  Xova  Act.  Reg.  Soc.  Scient.  L'psala  (3),  16;  Pfaff  and  Balch,  Joum. 
of  Exp.  Med..  1897;   Brand,  Pfliiger's  Arch..  90. 

*  Stadelmann,  Der  Icterus,  etc.,  Stuttgart,  1891. 


308  THE   LIVER. 

AlbertoniV  under  these  conditions  the  absolute  quantity  of  sohds  de- 
creases, while  the  relative  quantity  increases.  After  partaking  of  food  the 
secretion  increases  again.  The  statements  are  very  contradictory  in  regard 
to  the  time  necessary  after  partaking  of  food  before  the  secretion  reaches 
its  maximum.  After  a  careful  examination  and  compilation  of  all  the 
existing  statements  Heidenhain  ^  has  come  to  the  conclusion  that  in  dogs 
the  curve  of  rapidity  of  secretion  shows  two  maxima,  the  first  at  the  third 
to  fifth  hour  and  the  second  at  the  thirteenth  to  fifteenth  hour  after  par- 
taking of  food.  According  to  Barbera  the  time  when  the  maximum 
occurs  is  dependent  upon  the  kind  of  food.  With  carbohydrate  food  it 
is  two  to  three  hours,  after  protein  food  three  to  four  hours,  and  with  fat 
diet  it  is  five  to  seven  hours  after  feeding. 

According  to  the  older  statements,  the  proteins,  of  all  the  various  foods, 
cause  the  greatest  secretion  of  bile,  while  the  carbohydrates  diminish  the 
secretion,  or  at  least  excite  it  much  less  than  the  proteins.  This  coincides 
with  the  recent  observations  of  Barbera.^  The  authorities  are  by  no 
means  ageed  as  to  the  action  of  the  fats.  While  many  older  investigators 
have  not  observed  any  increase,  but  rather  the  reverse,  in  the  secretion  of 
bile  after  feeding  with  fats,  the  researches  of  Barbera  show  an  undoubted 
increase  in  the  secretion  of  bile  on  fat  feeding,  greater  even  than  after  car- 
l^ohydrate  feeding.  According  to  Rosenberg  olive-oil  is  a  strong  chola- 
gogue,  a  statement  which,  according  to  other  investigators — Mandelstamm, 
DoYON  and  Dufourt^ — is  not  sufficiently  proved. 

As  Barbera  has  shown,  a  close  relationship  exists  between  the  bile 
secretion  and  the  quantity  of  urea  formed,  as  an  increase  in  the  first  goes 
hand  in  hand  with  an  increase  of  the  latter.  The  bile  is,  therefore,  accord- 
ing to  him,  a  product  of  disassimilation,  whose  quantity  rises  and  falls  with 
the  degree  of  activity  of  the  liver. 

The  question  whether  there  exist  special  medicinal  bodies,  so-called 
cholagogues,  which  have  a  specific  excitant  action  on  the  secretion  of  bile 
has  been  answered  in  very  different  ways.  Many,  especially  the  older 
investigators,  have  observed  an  increase  in  the  bile  secretion  after  the  use 
of  certain  therapeutic  agents,  such  as  calomel,  rhubarb,  jalap,  turpentine, 


'  Lukjanow,  Zeitschr.  f.  pliysiol.  Chem.,  16;  Albertoni,  Recherches  sur  la  s6cr^tion 
biliaire,  Turin,   1893. 

^  Hermann's  Handb.,  5,  and  Stadelmann,  Der  Icterus,  etc. 

3  Centralbl.  f.  Physiol.,  12  and  16. 

*  Barbera,  Bull,  della  scienz.  med.  di  Bologna  (7),  5,  Maly's  Jahresber.,  24,  and 
Centralbl.  f.  Physiol.,  12  and  16;  Rosenberg,  Pfli'iger's  Arch.,  46;  Mandelstamm,  Ueber 
den  Einfluss  einiger  Arzneimittel  auf  Sekretion  und  Zusammensetzung  der  Galle  (Dis- 
sert. Dorpat,  1890);  Doyon  and  Dufourt,  Arch,  de  Physiol.  (5),  9.  In  regard  to  the 
action  of  various  foods  on  the  secretion  of  bile  see  also  Heidenhain,  1.  c. ;  Stadelmann, 
Der  Icterus;   and  Barbara,  1.  c. 


SECRETIOX   OF    BILE.  309 

olive-oil.  etc.;  while  others,  especially  the  more  recent  investigators,  have 
arrived  at  quite  opposite  results.  From  all  appearances  this  contradiction 
is  due  to  the  great  irregularity  of  the  normal  secretion,  which  might 
readily  cause  mistakes  in  tests  with  therapeutic  agents. 

Schiff's  view,  that  the  bile  absorbed  from  the  intestinal  canal  increases 
the  secretion  of  bile  and  hence  acts  as  a  cholagogue,  seems  to  be  a  positively 
proven  fact  by  the  investigations  of  several  experimenters.^  Sodiimi 
salicylate  is  also  perhaps  a  cholagogue  (Stadel:nl\xx,  Botox  and 
Dufouet). 

Acids,  and  especially,  imder  normal  conditions,  hydrochloric  acid,  seem 
to  be  physiological  excitants  for  bile  secretion.  According  to  Falloise 
and  Fleig  the  acids  act  upon  the  duodenum  and  the  upper  part  of  the 
jejunum,  and  the  action  is  brought  about  by  a  secretin  formation  similar 
to  the  action  of  acids  upon  the  secretion  of  pancreatic  juice  (see  Chapter 
IX).  According  to  Falloise  ~  chloral  hydrate  introduced  into  the  duo- 
denima  causes  a  secretion  of  bile  in  an  analogous  manner  by  the  aid  of  a 
special  chloral  secretin. 

The  bile  is  a  mixture  of  the  secretion  of  the  liver-cells  and  the  so-called 
mucus  which  is  secreted  by  the  glands  of  the  biliary  passages  and  by  the 
mucous  membrane  of  the  gall-bladder.  The  secretion  of  the  liver,  which 
is  generally  poorer  in  solids  than  the  bile  from  the  gall-bladder,  is  thin  and 
clear,  while  the  bile  collected  in  the  gall-bladder  is  more  ropy  and  viscous 
on  account  of  the  absorption  of  water  and  the  admixture  of  ''mucus,"  and 
cloudy  because  of  the  admixture  of  cells,  pigments,  and  the  like.  The 
specific  gra\'ity  of  the  bile  from  the  gall-bladder  varies  considerably,  being 
in  man  between  1.010  and  1.040.  Its  reaction  is  alkaline  to  litmus.  The 
color  changes  in  different  animals:  golden  yellow,  yellowish  brown,  olive- 
brown,  bro^Tiish  green,  grass-green,  or  bluish  green.  Bile  obtained  from 
an  executed  person  immediately  after  death  is  golden  yellow  or  yellow 
with  a  shade  of  bro-^Ti.  Still  cases  occur  in  which  fresh  human  bile  from 
the  gall-bladder  has  a  green  color.  The  ordinary  post-mortem  bile  has 
a  variable  color.  The  bile  of  certain  animals  has  a  peculiar  odor;  for 
example,  ox-bile  has  an  odor  of  musk,  especially  on  warming.  The  taste 
of  bile  is  also  different  in  different  animals.  Human  as  well  as  ox  bile  has 
a  bitter  taste,  with  a  sweetish  after-taste.  The  bile  of  the  pig  and  rabbit 
has  an  intensely  persistent  bitter  taste.  On  heating  bile  to  boiling  it  does 
not  coagulate.     It  contains  (in  the  ox)  only  traces  of  true  mucin,  and   its 

'  Schiff.  Pfliiger's  Arch..  3.  See  Stadelmann,  Der  Icterus,  and  the  dissertations  of 
his  pupils,  especially  Winteler,  '"  Experimentelle  Beitrage  ziu"  Frage  des  Kreislaufes 
der  Galle"  (Inaug.-Diss.  Dorpat,  1S92).  and  Gartner,  '•Experimentelle  Beitrage  zur 
Physiol,  und  Path,  der  Gallensekretion"  (Inaug.-Diss.  Jurjew,  1S93);  also  Stadehuann, 
"Ueber  den  Ivreislauf  der  Galle."  Zeitschr.  f.  Biologie.  34. 

'  Falloise,  Bull.  Acad.  Rov.  de  Belg.,  1903:    Fleig,  ibid..  1903. 


310  THE   LIVER. 

ropy  properties  depend,  it  seems,  chiefly  on  the  presence  of  a  nucleoalbumin 
similar  to  mucin  (Paijkull).  The  bile  from  the  animals  investigated 
by  Hammarsten  showed  a  similar  behavior.  Hammarsten  ^  has,  on  the 
contrary,  found  a  true  mucin  in  human  bile.  To  all  appearances  this 
mucin  originates  from  the  biliary  passages,  as  he  found  it  in  the  bile  flowing 
from  the  hepatic  duct,  and  also  because  the  mucous  membrane  of  the  gall- 
bladder, according  to  Wahlgren,^  does  not  in  man  secret  any  mucin,  but 
a  mucin-like  nucleoalbumin. 

The  specific  constituents  of  the  bile  are  bile-acids  combined  with  alkalies, 
bile-pigments,  and,  besides  small  quantities  of  lecithin  and  phospJmtides, 
cholesterin,  soaps,  neutral  fats,  urea,  ethereal  sulphuric  acid,  traces  of  con- 
jugated glvx;uronic  acids  and  mineral  substances,  chiefly  chlorides,  besides 
phosphates  of  calcium,  magnesium,  and  iron.     Traces  of  copper  also  occur. 

Bile-salts.  The  bile-acids  which  thus  far  have  best  been  studied  may 
be  divided  into  two  groups,  the  glycocholic  and  taurocholic  axid  groups.  As 
found  by  Hammarsten,^  a,  third  group  of  l)ile-acids  occurs  in  the  shark  and 
probably  also  in  other  animals.  These  are  rich  in  sulphur,  and  like  the 
ethereal  sulphuric  acids  they  split  off  sulphuric  acid  on  boiling  with  hydro- 
chloric acid.  All  glycocholic  acids  contain  nitrogen,  but  are  free  from 
sulphur  and  can  be  split  with  the  addition  of  water  into  glycocoU  (amino- 
acetic  acid)  and  a  nitrogen-free  acid,  a  cholic  acid.  All  taurocholic  acids 
contain  nitrogen  and  sulphur  and  are  split,  with  the  addition  of  water, 
into  taurine  (aminoethylsulphonic  acid)  and  a  cholic  acid.  The  reason 
for  the  existence  of  different  glycocholic  and  taurocholic  acids  depends 
on  the  fact  that  there  are  several  cholic  acids. 

The  conjugated  bile-acid  found  in  the  shark,  and  called  scymnol  sulphuric  add 
by  Hammarsten,  yields  as  cleavage  products  sulphuric  acid  and  a  non-nitrogenous 
substance,  scymnol  (CgiH^j^Og),  which  gives  the  characteristic  color  reactions  of 
cholic  acid. 

The  different  bile-acids  occur  in  the  bile  as  alkali  salts,  generally  the 
sodium  compounds,  even  in  sea-fishes,  although  this  is  contrary  to  the 
older  statements  (Zaxetti  ■*).  In  the  bile  of  certain  animals  we  find 
almost  solely  glycocholic  acid,  in  others  only  taurocholic  acid,  and  in 
other  animals  a  mixture  of  both  (see  further  on). 

All  alkali  salts  of  the  biliary  acids  are  soluble  in  water  and  alcohol,  but 
insoluble  in  ether.  Their  solution  in  alcohol  is  therefore  precipitated  by 
ether,  and  this   precipitate,  with  proper  care  in  manipulation,  gives,  for 

'  Paijkull,  Zeitschr.  f.  physiol.  Chem.,  12;    Hammarsten,  1.  c,  Nova  Act.  (3),  16, 
and  Ergebnisse  der  Physiol.,  Bd.  4. 
2  Maly's  Jahresber.,  32. 

'  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  24. 
^  See  Chem.  Centralbl.,  1903,  1,  180. 


BILE  ACIDS.  3il 

nearly  all  kinds  of  bile  thus  far  investigated,  rosettes  or  balls  of  fine  needles 
or  four-  to  six-sided  prisms  (Plattxer's  crystallized  bile).  Fresh  human 
bile  also  crj^stallizes  readily.  The  bile-acids  and  their  salts  are  optically 
active  and  dextrorotatory.  The  salts  of  the  different  bile-acids  act  some- 
what differently  towards  neutral  salts.  The  alkali  salts  of  the  ordinarj-  and 
best-studied  bile-acids  from  man,  ox,  and  dog  are,  according  to  Tengstrom,i 
precipitated  by  ammonium  and  magnesium  sulphates,  and  also,  in  pure 
form,  by  sodium  nitrate  and  sodium  chloride  (added  to  saturation).  Potas- 
sium and  sodium  sulphates  do  not  precipitate  them.  The  alkali  salts 
cannot  be  directly  precipitated  from  the  bile  by  NaCl,  on  account  of  the 
presence  of  bodies  retarding  precipitation,  among  which  we  find  oil-soaps. 

The  bile-acids  are  dissolved  by  concentrated  sulphuric  acid  at  the 
ordinar}'  temperature,  forming  a  reddish-yellow  liquid  which  has  a  beautiful 
green  fluorescence.  According  to  Pregl  an  oxidation  with  reduction  of 
the  sulphuric  acid  into  sulphur  dioxide  takes  place.  The  fluorescent 
substance  has  been  called  dehydrocholan  (see  below)  by  Pregl.^  On 
carefully  warming  with  concentrated  sulphuric  acid  and  a  little  cane-sugar, 
the  bile-acids  give  a  beautiful  cherrj'-red  or  reddish-violet  liquid.  Pet- 
tenkofer's  reaction  for  bile-acids  is  based  on  this  behavior. 

Pettenkofer's  test  for  bile-acids  is  performed  as  follows:  A  small 
quantity  of  bile  in  substance  is  dissolved  in  a  small  porcelain  dish  in  con- 
centrated sulphuric  acid  and  warmed,  or  some  of  the  liquid  containing  the 
bile-acids  is  mixed  with  concentrated  sulphuric  acid,  taking  special  care 
in  both  cases  that  the  temperature  does  not  rise  higher  than  60-70°  C.  Then 
a  10  per  cent  solution  of  cane-sugar  is  added,  drop  by  drop,  continually 
stirring  with  a  glass  rod.  The  presence  of  bile  is  indicated  by  the  produc- 
tion of  a  beautiful  red  liquid,  whose  color  does  not  disappear  at  the  ordinary 
temperature,  but  becomes  more  bluish  violet  in  the  course  of  a  day.  This 
red  liquid  shows  a  spectrum  with  two  absorption-bands,  the  one  at  F  and 
the  other  between  D  and  E,  near  E. 

This  extremely  delicate  test  fails,  however,  when  the  solution  is 
heated  too  high  or  if  an  improper  quantity — generally  too  much — of 
the  sugar  is  added.  In  the  last-mentioned  case  the  sugar  easily  car- 
bonizes and  the  test  becomes  brown  or  dark  bro-^Ti.  The  reaction  fails 
if  the  sulphuric  acid  contains  sulphurous  acid  or  the  lower  cfecides  of  nitro- 
gen. Many  other  substances,  such  as  proteins,  oleic  acid,  amyl  alcohol, 
and  morphine,  give  a  similar  reaction,  and  therefore  in  doubtful  cases 
the  spectroscopic  examination  of  the  red  solution  must  not  be  forgotten. 

Pettenkofer's  test  for  the  bile-acids  depends  essentially  on  the  fact 
that  furfurol  is  formed  from  the  sugar  by  the  sulphuric  acid,  and  this  body 
can  therefore  be  substituted  for  the  sugar  in  this  test  (Mylius).     Accord- 

•  Zeitschr.  f.  physiol.  Chem.,  41.  ^  Zeitschr.  f.  physiol.  Chem.,  45. 


312  THE   LIVER. 

ing  to  Mylius  and  v.  Udranszky  ^  a  1  p,  m.  solution  of  furfurol  should 
be  used.  Dissolve  the  bile,  which  must  first  be  purified  by  animal  charcoal, 
in  alcohol.  To  each  cubic  centimeter  of  alcoholic  solution  of  bile  in  a  test- 
tube  add  1  drop  of  the  furfurol  solution  and  1  c.c.  concentrated  sulphuric 
acid,  and  cool  when  necessary,  so  that  the  test  does  not  become  too  warm. 
This  reaction,  when  performed  as  described,  will  detect  2^  to  3V  rnilligram 
cholic  acid  (v.  Udranszky).  Other  modifications  of  Pettenkofer's 
test  have  been  proposed. 

Glycocholic  Acid.  The  constitution  of  the  glycocholic  acid  occurring 
in  human  and  ox  bile,  and  which  has  been  most  studied,  is  represented  by 
the  formula  C26H43NO6.  Glycocholic  acid  is  absent,  or  nearly  so,  in  the  bile 
of  carnivora.  On  boiling  with  acids  or  alkalies  this  acid,  which  is  anal- 
ogous to  hippuric  acid,  is  converted  into  cholic  acid  and  glycocoll. 

By  the  action  of  hydrazine  hydrate  upon  the  ethyl  ester  of  cholic  acid 
BoNDi  and  Mijller  ^  have  prepared  first  cholic-acid  hydrazide,  and  then, 
by  the  action  of  nitrous  acid  upon  this,  they  oljtained  the  cholic-acid  azide, 
C23H39O3CO.N3,  and  finally  from  this  last  in  alkaline  solution  with  glyco- 
coll they  synthetically  prepared  the  alkali  salt  of  glycocholic  acid,  at  the 
same  time  splitting  off  nitrogen. 

Glycocholic  acid  crystallizes  in  fine,  colorless  needles  or  prisms.  It  is 
soluble  with  difficulty  in  water  (in  about  300  parts  cold  and  120  parts  boil- 
ing water),  and  is  easily  precipitated  from  its  alkali-salt  solution  by  the 
addition  of  dilute  mineral  acids.  It  is  readily  soluble  in  strong  alcohol,  but 
with  great  difficulty  in  ether.  The  solutions  have  a  bitter  but  at  the  same 
time  sweetish  taste.  The  acid  melts  at  138-140°  C.  (Medvedew^).  The 
salts  of  the  alkalies  and  alkaline  earths  are  soluble  in  alcohol  and  water. 

The  solution  of  the  alkali  salt  in  water  can  be  salted  out  by  NaCl,  but 
not  by  KCl.  The  salts  of  the  heavy  metals  are  mostly  insoluble  or  soluble 
with  difficulty  in  water.  The  solution  of  the  alkali  salts  in  water  is  pre- 
cipitated by  sugar  of  lead,  cupric  and  ferric  salts,  and  silver  nitrate. 

Glycocholeic  Acid  is  a  second  glycocholic  acid,  first  isolated  by  Wahl- 
GREN"*  from  ox-bile,  and  has  the  formula  C26H43NO5  or  C27H45NO5.  This 
acid,  which  on  hydrolytic  cleavage  yields  glycocoll  and  choleic  acid,  has 
also  been  detected  in  human  bile  and  the  bile  of  the  musk-ox  (Hammar- 

STEN  ^). 

Glycocholeic  acid  may,  like  glycocholic  acid,  crystallize  in  tufts  of 
fine  needles,  but  is  often  obtained  as  short  thick  prisms.  It  is  much  more 
insoluble  in  water,  even  on  boiling,  than  glycocholic  acid,  and  it  melts  at 

»  Mylius,  Zeitschr.  f.  physiol.  Chem.,  11;  v.  Udranszky,  ihid.,  12. 

2  Zeitschr.  f.  physiol.  Chem.,  47. 

^Centralbl.  f.  Physiol.,  14. 

*  Zeitschr.  f.  physiol.  Chem.,  36. 

«/bzd..43. 


GLYCOCHOLEIC   AND  TAUROCHOLIC    ACIDS.  313 

175-176°  C.  The  alkali  salts  are  soluble  in  water,  have  a  pure  bitter 
taste,  and  are  more  readily  precipitated  by  neutral  salts  (NaCl)  than  the 
glycocholates.  The  solution  of  the  alkali  salts  is  not  only  precipitated 
by  the  salts  of  the  heavy  metals,  but  also  by  the  salts  of  barium,  calcium 
and  magnesium. 

The  preparation  of  the  pure  glycocholic  acids  may  be  performed  in 
several  ways.  The  bile,  which  has  been  freed  from  mucus  by  means  of 
alcohol  and  the  alcohol  removed  by  evaporation,  may  be  precipitated  by 
a  solution  of  lead  acetate.  The  precipitate  is  then  decomposed  by  a  soda 
solution  and  heat,  evaporated  to  dryness,  and  the  residue  extracted  with 
alcohol,  which  dissolves  the  alkali  glycocholate.  The  alcohol  is  distilled 
from  the  filtered  solution  and  the  residue  dissolved  in  water;  this  solution 
is  now  decolorized  by  animal  charcoal  and  the  glycocholic  acid  precipitated 
from  the  solution  by  the  addition  of  a  dilute  mineral  acid.  The  mixture 
of  the  two  glycocholic  acids  is  freed  from  mineral  acid  by  carefully  washing 
with  water,  and  then  is  boiled  with  water,  when  the  glycocholic  acid  dissolves 
and  may  be  obtained  from  the  filtrate  as  crystals  on  cooling.  The  glyco- 
choleic  acid  with  some  transformed  glycocholic  acid  (paraglycocholic  acid) 
remains  undissolved  and  may  be  purified  by  converting  it  into  the  insoluble 
barium  salt.  If  we  do  not  care  for  the  obtainment  of  pure  glycocholeic  acid 
but  want  only  the  pure  glycocholic  acid,  then  the  decolorization  with  animal 
charcoal  can  be  omitted.  If  the  bile  is  rich  in  glycocholic  acid,  we  can 
treat  the  mucus-free  bile,  according  to  Hf  fner's  ^  method,  with  ether  and 
hydrochloric  acid,  when  the  glycocholic  acid  crystallizes  out  in  abundance. 
The  reader  is  referred  to  more  exhaustive  works  for  other  methods  of  prepa- 
ration. 

Hyoglycocholic  Acid,  CojH^gNOs,  is  the  crystalline  glycocholic  acid  obtained 
from  the  bile  of  the  pig.  It  is  very  insoluble  in  water.  The  alkali  salts,  whose 
solutions  have  an  intensely  bitter  taste,  without  any  sweetish  after-taste,  are 
precipitated  by  CaCb,  BaCb,  and  MgCb,  and  may  be  salted  out  like  a  soap  by 
Na2S04  when  added  in  sufficient  quantity.  By  precipitation  with  NaCl  in  such 
quantity  that  the  precipitate  redissolves  on  warming,  Hammarsten  ^  has  obtained 
the  alkali  salt  as  macroscopic  crystals  on  cooling.  Besides  this  acid  there  occurs 
in  the  bile  of  the  pig  still  another  glycocholic  acid  (Jolin  ^). 

The  glycocholate  in  the  bile  of  the  rodent  is  also  precipitated  by  the  above- 
mentioned  earthy  salts,  but  cannot,  like  the  corresponding  salt  in  human  or  ox 
bile,  be  directly  precipitated  on  saturating  with  a  neutral  salt  (Na2S04).  Guano 
bile-acid  possibly  belongs  to  the  glycocholic-acid  group,  and  is  found  in  Peruvian 
guano,  but  has  not  been  thoroughly  studied. 

Taurocholic  Acid.  This  acid,  which  is  found  in  the  bile  of  man,  car- 
nivora,  oxen,  and  a  few  other  herbivora,  such  as  sheep  and  goats,  has  the 
constitution  C26H45NSO7.  On  boiling  with  acids  and  alkalies  it  splits  into 
cholic  acid  and  taurine.  Taurocholic  acid  has  also  been  prepared  syntheti- 
cally by  BoNDi  and  Mijller,  using  the  same  method  as  they  used  for  glyco- 
cholic acid. 

*  Journ.  f.  prakt.  Chem.,  1874. 

^  Not  published. 

^  Zeitschr.  f.  physiol.  Chem.,  12  and  13. 


314  THE   LIVER. 

Taurocholic  acid  can  be  readily  obtained,  by  the  method  suggested 
by  Hammarsten/  as  groups  of  fine  needles  or  as  beautiful  prisms  on 
slow  crystallization.  The  crystals  do  not  change  in  the  air,  but  they 
decompose  above  100°.  They  are  soluble  in  alcohol  but  insoluble  in  ether, 
benzene,  and  acetone.  Taurocholic  acid  is  very  soluble  in  water,  and  the 
solution  has  a  very  sweet  taste,  with  only  a  slight  bitter  taste.  It  can  hold 
the  difficultly  soluble  glycocholic  acid  in  solution.  This  is  the  reason  why 
a  mixture  of  glycocholate  with  a  sufficient  quantity  of  taurocholate,  which 
often  occurs  in  ox-bile,  is  not  precipitated  by  a  dilute  acid.  Its  salts  are, 
as  a  rule,  readily  soluble  in  water,  and  the  solutions  of  the  alkali  salts  are 
not  precipitated  by  copper  sulphate,  silver  nitrate,  or  sugar  of  lead.  Basic 
lead  acetate  gives,  on  the  contrary,  a  precipitate  which  is  soluble  in  boiling 
alcohol.  The  alkali  salts  are  not  only  precipitated  from  their  solution  by 
the  same  neutral  salts  that  precipitate  glycocholic  acid,  but  also  by  potas- 
sium chloride,  and  by  sodium  and  potassium  acetates. 

Taurocholic  acid  is  most  simply  prepared  from  a  glycocholic-acid-free 
bile  or  from  one  very  poor  in  this  acid,  such  as  fish-  or  dog-bile.  From 
ox-bile  it  can  be  prepared  by  first  precipitating  the  glycocholic  acid  with 
alum  and  then  repeatedly  precipitating  the  filtrate  with  ferric  chloride 
(according  to  Tengstrom).  From  this  filtrate  the  taurocholate  is  precip- 
itated by  saturating  with  NaCl,  the  precipitate  pressed  and  freed  from 
NaCl  by  dissolving  in  alcohol,  and  as  a  powder,  or  dissolved  in  a  little  alcohol, 
is  decomposed  by  alcohol  containing  hydrochloric  acid.  The  acid  is  pre- 
cipitated from  the  filtrate  by  ether.  The  taurocholic  acid  can  be  repeatedly 
recrystallized  by  solution  in  alcohol  containing  water  and  the  careful 
addition  of  ether. 

Taurocholeic  Acid  is  a  second  taurocholic  acid,  detected  by  Hammar- 
STEN  in  dog-bile  and  isolated  by  Gullbring  ^  from  ox-bile,  and  has  the 
formula  C26H45NSO6  or  C27H47NSO6.  Thus  far  it  has  been  obtained  only 
in  the  amorphous  form.  It  is  readily  soluble  in  water,  and  has  a  disagreeably 
bitter  taste.  It  is  also  readily  soluble  in  alcohol,  but  insoluble  in  ether, 
acetone,  chloroform,  and  benzene.  The  alkali  salt,  soluble  in  water,  can  be 
salted  out  by  NaCl  as  a  pasty  mass.  The  solutions  of  the  salts  can  be 
precipitated  by  ferric  chloride.  The  cleavage  products  are  taurine  and 
choleic  acid. 

For  the  preparation  of  taurocholeic  acid  it  is  best  to  use  dog-bile  which 
is  first  precipitated  by  ferric  chloride.  The  precipitate  contains  the  acid, 
while  the  filtrate  can  be  used  for  the  obtainment  of  taurocholic  acid  by 
saturating  with  NaCl.  The  iron  precipitate  is  converted  into  the  alkali 
salt   by  sodium    carbonate,  and    is    decomposed    by  alcohol    containing 


*  Zeitschr.  f.  physiol.  Chem.,  43. 

*  Hammarsten,  Zeitschr.  f.  physiol.  Chera.,  43;  Gullbring,  ibid.,  45. 


CHOLIC  ACID.  315 

hydrochloric  acid,  and  then  precipitated  by  ether.  I'he  amorphous  acid 
which  separates  is  purified  from  alcohohc  solution  by  precipitation  with 
ether.  In  preparing  taurochohc  acid  from  ox-bile,  the  taurocholeic  acid, 
which  is  readily  soluble  in  alcohol-ether,  remains  in  the  alcohol-ether  on 
the  proper  addition  of  ether.  This  crude  acid  as  alkali  salt  is  freed  from 
taurocholic  acid  by  precipitation  with  ferric  chloride,  then  again  converted 
into  alkali  salt,  decomposed  wdth  acid  in  alcohol,  precipitated  by  ether,  and 
purified  (Gullbrixg). 

Cheno-taurocholic  Acid.  This  is  the  most  essential  acid  of  goose-bile  and  has 
the  formula  C29H4  XSOg.  This  acid,  though  Uttle  studied,  is  amorphous  and  solu- 
ble in  water  and  alcohol. 

The  taurocholic  acids  differ  from  the  glycocholic  acids  in  being  readily 
soluble  in  water.  In  the  bile  of  the  walrus,  on  the  contrary,  a  relatively 
insoluble,  readily  cr}-stallizable  taurocholic  acid  occurs  which  can  be  pre- 
cipitated from  the  solution  of  the  alkali  salts  by  the  addition  of  mineral 
acids,  similar  to  glycocholic  acid  (Ha^hlirstex  ^). 

As  repeatedly  mentioned  above,  the  two  bile-acids  split  on  boiling  with 
acids  or  alkalies  into  non-nitrogenous  cholic  acids  and  glycocoll  or  taurine. 
Of  the  various  cholic  acids  the  following  have  been  best  studied. 

Cholic  Acid  or  Cholalic  Acid.  The  ordinary  cholic  acid  obtained  as  a 
decomposition  product  of  human  and  ox  bile,  which  occurs  regularly  in 
the  contents  of  the  intestine  and  in  the  urine  in  icterus,  has.  according  to 
Strecker  and  nearly  all  recent  investigators,  the  constitution  C24H4o05  = 

(  CHOH 
t'2oH3i  ■<  (CHoOH).,.     According  to  Mylius.^  cholic  acid  is  a  monobasic 
(  COOH 

alcohol-acid  wdth  one  secondary  and  two  primary  alcohol  groups.  CuR- 
Tius  3  has  sho-uTi  by  preparing  the  cholamine,  C23H39O3XH2,  from  the 
above-mentioned  (p.  312)  cholic-acid  azide,  with  cholic-acid  urethane  as  an 
intermediary  step,  that  the  carboxyl  group  is  not  immediately  connected 
with  the  CHOH  group,  but  is  combined  with  the  chief  nucleus  without 
the  neighboring  secondary  alcohol  group.  On  oxidation  it  first  yields 
dchydrocholic  acid,  C24H34O5  (HAMiLiRSTEx).  On  further  oxidation 
hilianic  acid,  C24H34O8  (Cleve),  is  obtained,  or.  more  correctly,  according 
to  Lassar-Cohx  and  Pregl,  a  mixture  of  bilianic  and  isohilianic  acids. 
On  oxidation,  bilianic  acid  yields  cilianic  acid  (Lassar-Cohn),  whose 
formula,  according  to  Pregl,  is  C20H28O8.  On  stronger  oxidation  it  yields 
cholesterinic  acid,  which  has  not  been  carefully  studied,  and  finally  phthalic 
acid,  as  maintained  by  Senkowski,  but  not  substantiated  by  Bulnheim 

•  Not  published. 

*  The  important  researches  of  Strecker  on  the  bile-acids  may  be  found  in  Annal.  d. 
Chem.  u.  Pharm.,  65,  67,  and  70;  Mylius,  Ber.  d.  deutsch.  chem.  Gesellsch.,  19. 

'  Ibid.,  39. 


316  THE  LIVER. 

or  Pregl.i  On  reduction  (in  putrefaction)  cholic  acid  may  yield  desoxy- 
cholic  acid,  C24H40O4  (Mylius).  On  reduction  with  hydriodic  acid  and  red 
phosphoruSj  Pregl  obtained  a  product  which  he  considers  as  a  mono-car- 

(CH2 
boxvlic  acid,  with  the  formula  C20H31  ]  (CH3)2.     Senkowski  has  obtained 

(  COOH 

an  acid  with  the  formula  C24H40O2,  cholylic  add,  on  the  reduction  of  tlie 
anhydride  .2 

As  above  mentioned,  Pregl  ^  has  obtained,  by  the  action  of  concentrated 
sulphuric  acid  upon  cholic  acid,  a  fluorescent  substance  which  he  calls 
dehydrocholon.  This  is  produced  by  oxidation,  and  at  the  same  time,  water 
is  eliminated.  It  has  probably  the  formula  C24H38O.  Dehydrocholon  is 
nitrated  by  nitric  acid,  while  the  cholic  acid  is  not.  From  this  behg-vior, 
as  well  as  from  the  determination  of  the  molecular  refraction  and  disper- 
sion of  both  bodies,  Pregl  finds  it  probable  that  cholic  acid  belongs  to 
the  hydrated  carbocyclic  compounds. 

Cholic  acid  crystallizes  partly  in  rhombic  plates  or  prisms  with  1 
molecule  of  water  and  partly  in  larger  rhombic  tetrahedra  or  octahedra 
with  1  molecule  of  alcohol  of  crystallization  (Mylius).  These  crystals 
become  quickly  opaque  and  porcelain-white  in  the  air.  They  are  quite 
insoluble  in  water  (in  4000  parts  cold  and  750  parts  boiling),  rather  soIu])le 
in  alcohol,  but  soluble  with  difficulty  in  ether.  The  amorphous  cholic  acid 
is  less  insoluble.  The  solutions  have  a  bitter-sweetish  taste.  The  crys- 
tals lose  their  alcohol  of  crystallization  only  after  a  lengthy  heating  to 
100-120°  C.  The  acid  free  from  water  and  alcohol  melts  at  195°  C.  Accord- 
ing to  BoNDi  and  MIjller  the  melting-point  of  the  perfectly  pure  acid  is 
198°.     It  forms  a  characteristic  blue  compound  with  iodine  (Mylius). 

The  alkali  salts  are  readily  soluble  in  water,  but  when  treated  with  a 
concentrated  caustic  or  carbonated  alkali  solution  may  be  separated  as  an 
oily  mass  which  becomes  crystalline  on  cooling.  The  alkali  salts  are  not 
readily  soluble  in  alcohol,  and  on  the  evaporation  of  the  alcohol  they  may 
crystallize.  The  specific  rotatory  power  of  the  sodium  salt  is  (a)D  = 
+  31.4°.*  The  watery  solution  of  the  alkali  salts,  when  not  too  dilute,  is 
precipitated  immediately  or  after  some  time  by  sugar  of  lead  or  by  barium 
chloride.  The  barium  salt  crystallizes  in  fine,  silky  needles,  and  it  is  rather 
insoluble  in  cold,  Init  somewhat  easilv  solul^le  in  warm  water.     The  barium 


*  Hammarsten,  Ber.  d.  deutsch.  chem.  Gesellsch.,  14;  Cleve,  Bull.  Soc.  chim.,  35; 
Lassar-Cohn,  Ber.  d.  d.  chem.  Gesellsch.,  32;  Pregl,  Wien.  Sitzungsber.,  Ill,  1902;  Sen- 
kowski, Monatshefte  f.  Chem.,  17;  Bulnheim,  Zeitschr.  f.  physiol.  Chem.,  25,  in  which 
the  literature  on  cholesterinic  acid  may  be  found. 

^  Mylius,  1.  c;   Pregl,  Pfliiger's  Arch.,  71;   Senkowski,  Monatshefte  f.  Chem.,  19. 

*  Zeitschr.  f.  physiol.  Chem.,  45. 

*  See  Vahlen,  Zeitschr.  f.  physiol.  Chem.,  21. 


CHOLEIC   ACID.  317 

salt,  as  well  as  the  lead  salt  which  is  insoluble  in  water,  is  soluble  in  warm 
alcohol. 

Choleic  Acid  (C25H42O4,  Latschinoff)  is  another  cholic  acid  which, 
according  to  Lassar-Cohn/  has  the  formula  C24H40O4.  This  acid,  which 
occurs  in  varjdng  but  always  small  quantities  in  ox-bile,  yields  dehydro^ 
choleic  acid,  C24H34O4,  and  then  cholanic  acid,  C24H34O7,  and  isocholanic 
acid  on  oxidation. 

Choleic  acid  crystallizes  when  free  from  water  in  hexagonal,  vitreous 
prisms  with  pointed  ends,  melting  at  185-190°  C.  The  crystalline  acid  con- 
taining water  melts  at  135-140°  C.  (Latschinoff).  The  acid  dissolves 
in  water  with  difficulty  and  is  also  relatively  difficultly  soluble  in  alcohol. 
It  has  an  intensely  bitter  taste  and  gives  the  ]\Iylius  iodine  reaction  for  cholic 
acid.  The  specific  rotation  is  (a:)D= +48.87°  (Vahlen).  The  barium 
salt  which  crystallizes  from  the  hot  alcoholic  solution  as  spherical  aggre- 
gations of  radial  needles  is  more  difficulth"  soluble  in  water  than  the  cor- 
responding cholate. 

The  relation  of  choleic  acid  to  desoxycholic  acid  is  not  kno^n. 
According  to  Latschinoff  and  Lassar-Cohn  both  acids  are  identical, 
w^hile  Pregl,2  on  the  contrary,  claims  that  desoxycholic  acid  is  more 
readily  soluble  in  water  and  when  anhydrous  has  a  melting-point  of  172- 
173°.  According  to  the  ordinary  views  the  desoxj'-cholic  acid  is  formed 
from  cholic  acid  by  reduction.  Ekbom  ^  could  not  substantiate  this 
statement.  On  using  perfectly  pure  cholic  acid  he  was  able  to  regain 
nearly  quantitatively  after  the  action  of  metallic  sodium  on  the  alcoholic 
solution  of  the  acid  or  of  zinc  and  alkali.  By  treatment  with  zinc  and  acetic 
acid  a  reaction  took  place,  but  the  product  was  a  mixture  of  mono-  and 
diacetyl  derivatives.  This  indicates  that  desox3'cholic  acid  is  isomeric  with 
an  acid  preformed  in  the  bile,  either  choleic  acid  or  possibly,  as  Pregl  has 
shown,  an  acid  isomeric  therewith.  The  observation  of  Pregl  that 
desoxycholic  acid,  like  choleic  acid,  yields  dehydrocholeic  acid  and  cholanic 
acid  as  oxidation  products,  stands  in  close  connection  with  such  an  assump- 
tion, but  makes  the  formation  of  desoxycholic  acid  from  cholic  acid  by 
reduction  very  improbable. 

Both  cholic  acids  are  best  prepared  from  ox-bile  which  has  been  boiled 
for  twenty-four  hours  with  baryta-water  or  caustic  soda.  According  to 
Mylius,^  boil  the  bile  for  twenty-four  hours  with  five  times  its  weight  of  a 
30  per  cent  caustic-soda  solution,  replacing  the  water  lost  by  evaporation. 

'  Latschinoff,  Ber.  d.  deutsch.  chem.  Gesellsch.,  18  and  20;  Lassar-Cohn,  ibid.,  26, 
and  Zeitschr.  f.  physiol.  Chem.,  I".     See  also  Vahlen.  Zeitschr.  f.  physioi!  Chem.,  23. 

'  Wien.  Sitzungsber.,  111.  Math.  Naturw.  Kl.  1902;  Latschinoff,  1.  "..;  Lassar- 
Cohn,  1.  c.     See  also  Myhus,  Ber.  d.  d.  chem.  Gesellsch.,  19. 

^  Unpublished  investigation. 

*  Zeitschr.  f.  physiol.  Chem.,  12. 


318  THE  LIVER. 

Now  saturate  the  liquid  with  CO2  and  evaporate  nearly  to  dryness.  The 
residue  is  extracted  with  9G  per  cent  alcohol  and  this  alcohohc  extract 
diluted  with  water  until  it  contains  at  the  most  20  per  cent  alcohol;  it  is 
then  completely  precipitated  with  a  BaCl2  solution.  The  precipitate, 
which  contains  besides  fatty  acids  also  the  choleic  acid,  is  filtered,  and 
the  cholic  acid,  contaminated  with  choleic  acid,  is  precipitated  from  the 
filtrate  by  hydrochloric  acid.  After  the  cholic  acid  has  gradually  crys- 
tallized out  it  is  repeatedly  recrj'stallized  from  alcohol  or  methyl  alcohol. 
According  to  Boxdi  and  Mlller,!  perfectly  pure  cholic  acid  having  a 
melting-point  of  198°  can  be  obtained  by  boiling  the  impure  acid  for  four 
hours  with  10  per  cent  caustic  soda,  reprecipitating  with  hydrochloric  acid, 
and  recrystallizing  from  .alcohol. 

Choleic  acid  may  l)e  obtained  from  the  above-mentioned  l^arium  pre- 
cipitate by  first  converting  the  barium  salt  into  sodium  salt  by  sodium  car- 
bonate, then  fractionally  precipitating  the  fatty  acids  by  barium  acetate, 
separating  the  choleic  acid  from  the  filtrate  by  hydrochloric  acid  and 
recrystallizing  several  times  from  glacial  acetic  acid. 

Pregl  2  has  suggested  a  somewhat  different  but  simpler  method  for 
preparing  cholic  acid  and  obtaining  the  desoxycholic  acid  from  ox-bile.  In 
regard  to  this  as  well  as  other  methods  of  preparation  we  must  refer  to 
the  original  communications  and  to  other  handbooks. 

Fellic  Acid,  C23H40O4,  is  a  cholic  acid,  so  called  by  Schotte:^,  which 
he  obtained  from  human  bile,  along  with  the  ordinary  acid.  This  acid  is 
crystalline,  is  insoluble  in  water,  and  yields  barium  and  magnesium  salts 
which  are  very  insoluble.  It  does  not  respond  to  Pettexkofer's  reaction 
easily  and  gives  a  more  reddish-blue  color. 

The  conjugate  acids  of  human  bile  have  not  been  sufficiently  investi- 
gated. To  all  appearances  human  bile  contains  under  different  circum- 
stances various  conjugate  bile-acids.  In  some  cases  the  bile-salts  of  human 
bile  are  precipitated  l)y  BaCl2  and  in  others  not.  According  to  the  state- 
ments of  Lassar-Cohx^  three  cholic  acids  may  l^e  prepared  from  human 
bile,  namely,  ordinary  cholic  acid,  choleic  acid,  and  fellic  acid. 

Lithofellic  Acid,  CjoHagO^,  is  the  acid  related  to  cholic  acid  which  occurs  in 
the  oriental  bezoar  stones,  which  is  insoluble  in  water,  comparatively  easUy  solu- 
ble in  alcohol,  but  only  slightly  soluble  in  ether.* 

The  hyo-glycocholic  and  cheno-taurocholic  acids,  as  well  as  the  gh'co- 
cholic  acid  of  the  bile  of  rodents,  yield  corresponding  cholic  acids.  This 
seems  to  be  the  case  also  with  the  glycocholic  acid  of  the  hippopotamus- 
bile,  which  stands  very  close  to  the  pig-bile  (Hammarstex  ^).     In  the  polar 

'  Zeitschr.  f.  physiol.  Chem.,  47. 
"1.  c,  Wien.  Sitzungsber. 

^  Schotten,  Zeitschr.  f.  physiol.  Chera.,  11;  Lassar-Cohn,  Ber.  d.  deutsch.  chem. 
Gesellsch.,  27. 

*  See  Jiinger  and  Klages,  Ber.  d.  deutsch.  ch2m.  Gesellsch.,  28  (older  hterature). 
'  Investigations  not  published. 


BILE   PIGMENTS.  319 

bear  a  third  cholic  acid  exists  besides  cholic  and  choleic  acids.  It  is  called 
ursochokic  acid,  C19H30O4  or  C18H28O4  (Hammarsten  ^).  The  bile  of 
other  animals  (walrus,  sea-dog)  contains  special  cholic  acids  (Haivimar- 
STEN  2), 

On  boiling  with  acids,  on  putrefaction  in  the  intestine,  or  on  heating, 
cholic  acids  lose  water  and  are  converted  into  anhydrides,  the  so-called 
dyslysins.  The  dj'slysin,  C24H36O3,  corresponding  to  ordinary  cholic  acid, 
which  occurs  in  faeces,  is  amorphous,  insoluble  in  water  and  alkalies. 
Choloidic  acid,  C24H38O4,  is  called  the  first  anhydride  or  an  intermediary 
product  in  the  formation  of  dyslysin.  On  boiling  dyslj'sins  with  caustic 
alkali  they  are  reconverted  into  the  corresponding  cholic  acids. 

The  Detection  of  Bile-acids  in  Aximal  Fluids.  To  obtain  the 
bile-acids  pure  so  that  Pettexkofer's  test  can  be  applied  to  them,  the 
protein  and  fat  must  first  be  removed.  The  protein  is  removed  by  making 
the  liquid  first  neutral  and  then  adding  a  great  excess  of  alcohol,  so  that 
the  mixture  contains  at  least  85  vols,  per  cent  of  water-free  alcohol.  Now 
filter,  extract  the  precipitated  protein  with  fresh  alcohol,  unite  all  filtrates, 
distil  the  alcohol,  and  evaporate  to  drj'ness.  The  residue  is  completely 
exhausted  with  strong  alcohol,  filtered,  and  the  alcohol  entirely  evaporated 
from  the  filtrate.  The  new  residue  is  dissolved  in  water,  and  filtered  if 
necessary,  and  the  solution  precipitated  by  basic  lead  acetate  and  am- 
monia. The  washed  precipitate  is  dissolved  in  boiling  alcohol,  filtered 
while  warm,  and  a  few  drops  of  soda  solution  added.  Then  evaporate  to 
dryness,  extract  the  residue  with  absolute  alcohol,  filter,  and  add  an  excess 
of  ether.  The  precipitate  now  formed  may  be  used  for  Pettexkofer's 
test.  It  is  not  necessary  to  wait  for  crystallization;  but  one  must  not 
consider  the  crystals  which  form  in  the  liquid  as  being  positively  crystal- 
lized bile.  It  is  also  possible  for  needles  of  alkali  acetate  to  be  formed. 
For  the  detection  of  bile-acids  in  urine  see  Chapter  XV. 

Bile-pigments.  The  bile-coloring  matters  known  thus  far  are  relatively 
numerous,  and  in  all  probability  there  are  still  more  of  them.  Most  of  the 
known  bile-pigments  are  not  found  in  the  normal  bile,  but  occur  either  in 
postmortem  bile  or  principally  in  the  bile  concrements.  The  pigments  which 
occur  imder  physiological  conditions  are  the  reddish-yellow  bilirubin,  the 
green  biliverdin,  and  sometimes  also  urobilin  (and  urobilinogen)  or  a  closely 
related  pigment.  The  pigments  found  in  gall-stones  are  (besides  the 
bilirubin  and  biliverdin)  choleprasin,  bilijuscin,  biliyrasin,  bilihumin, 
bilicyanin  (and  choletelin?) .  Besides  these,  others  have  been  noticed 
in  human  and  animal  bile  by  various  observers.  The  two  above-men- 
tioned physiological  pigments,  bilirubin  and  biliverdin,  are  those  which 
serve  to  give  the  golden-yellow  or  orange-yellow  or  sometimes  greenish 
color  to  the  bile;   or  when,  as  is  most  frequently  the  case  in  ox-bile,  the 

'  Zeitschr.  f.  physiol.  Chem.,  3(5.  -  Investigations  not  published. 


320  THE    LIVER. 

two  pigments  are  present  in  the  bile  at  the  same  time,  they  produce  the 
different  shades  between  reddish  brown  and  green. 

Bilirubin.  This  pigment  has  the  formula  C16H18N2O3,  or  according 
to  Orndorff  and  Teeple  1  more  correctly  C32H36N4O6,  and  is  designated 
by  the  names  cholepyrrhix,  biliph.ein,  bilifulvin,  and  ilematoidIxN. 
It  occurs  chiefly  in  the  gall-stones  as  calcium  bilirubin.  Bilirubin  is  present 
in  the  liver-bile  of  all  vertebrates,  and  in  the  bladder-bile  especially  in 
man  and  carnivora;  sometimes,  however,  the  latter  may  have  a  green  bile 
when  fasting  or  in  a  starving  condition.  It  occurs  also  in  the  contents 
of  the  small  intestine,  in  the  blood-serum  of  the  horse,  in  old  blood  extrav- 
asations (as  hsematoidin),  and  in  the  urine  and  the  yellow-colored  tissue 
in  icterus.  It  is  converted  into  hydrobilirubin,  C32H40N4O7  (Maly),  by 
hydrogen  in  a  nascent  state,  and  then  shows  great  similarity  to  the  urinary 
pigment,  urobilin,  as  well  as  to  stercobilin  found  in  the  contents  of  the 
intestine  (Masius  and  Vanlair^).  On  careful  oxidation  bilirubin  yields 
biliverdin  and  other  coloring-matters  (see  below). 

Bilirubin  is  derived  from  the  blood-pigment.  It  has  the  same  per- 
centage composition  as  hsematoporphyrin,  and  like  hsematin  it  yields 
hsematinic-acid  imide  as  an  oxidation  product  (Kxjster).  On  reduction 
v\'ith  zinc  powder  or  with  nascent  HI,  it  yields  hgemopyrrol  according  to 
Orndorff  and  Teeple.^ 

Bilirubin  is  sometimes  amorphous  and  sometimes  crystalline.  The  amor- 
phous bilirubin  is  a  reddish-yellow  or  reddish-brown  powder;  the  crystals 
have  a  reddish-yellow,  reddish-brown,  or  more  reddish  color,  and  sometimes 
they  have  nearly  the  color  of  crystalline  chromic  acid.  The  crystals,  which 
can  easily  be  obtained  by  allowing  a  solution  of  bilirubin  in  chloroform  to 
evaporate  spontaneously,  are  reddish-yellow,  rhombic  plates,  whose  obtuse 
angles  are  often  rounded.  On  crystallizing  from  hot  dimethylaniline  it 
forms  on  cooling  broad  columns  with  both  ends  sharply  cut  (KiisTER*). 
On  dissolving  in  chloroform  both  kinds  of  crystals  are  converted  into  long 
needles  or  whetstones. 

Bilirubin  is  insoluble  in  water,  behaves  like  an  acid,  and  occurs  in 
animal  fluids  as  soluble  alkali  bilirubin.  It  is  very  slightly  soluble  in  ether, 
benzene,  carbon  disulphide,  amyl  alcohol,  fatty  oils,  and  glycerine.  It  is 
somewhat  more  soluble  in  alcohol.  In  cold  chloroform  it  dissolves  with 
difficulty,  and  is  much  more  readily  soluble  in  warm  chloroform.  Its  solu- 
bility varies,  and  supersaturated  solutions  are  readily  formed  (Orndorff 
and  Teeple).     The  varying  solubility  of  bilirubin  in  chloroform  depends, 

'  Salkowski's  Festschrift,  Berlin,  1904. 

-  Maly,  Wien.  Sitzungsber.,  57,  and  Annal.  d.  Chem.,  163;   Masius  and  Vanlair, 
Centralbl.  f.  d.  med.  Wissensch.,  1871,  369. 
M.  c. 
*  Ber.  d.  d.  chem.  Gesellsch.,  30  and  35,  and  Zeitschr.  f.  physiol.  Chem.,  47. 


BILIRUBIX.  321 

according  to  Kuster.  on  the  fact  that  in  its  preparation  derivatives  which 
are  readily  soluble  and  contain  chlorine  or  other  transformation  products  are 
formed,  or  perhaps  the  bilirubin  goes  over  into  polymeric  modifications  have 
ing  different  solubilities.  In  cold  dimethylaniline  it  dissolves  in  the  propor- 
t  on  of  1 :  100,  and  in  hot  dimethylaniline  much  more  readily.  Its  solutions 
show  no  absorption-bands,  but  only  a  continuous  absorption  from  the  red 
to  the  violet  end  of  the  spectrum,  and  they  have,  even  on  diluting  greatly 
(.1:500  000),  in  a  layer  1.5  cm.  thick  a  decided  yellow  color.  If  a  dilute 
solution  of  alkali  bilirubin  in  water  is  treated  with  an  excess  of  ammonia 
and  then  with  a  zinc-chloride  solution,  the  liquid  is  first  colored  deep  orange 
and  then  gradually  olive-broA\Ti  and  then  green.  This  solution  first  gives 
a  darkening  of  the  ^■iolet  and  blue  part  of  the  spectrum  and  then  the  bands 
of  alkaline  cholecyanin  (see  below),  or  at  least  the  binds  of  this  pigment 
in  the  red  between  C  and  D,  close  to  C.  This  is  a  good  reaction  for  bilirubin. 
The  compounds  of  bilirubin  with  alkalies  are  insoluble  in  chloroform,  and 
bilirubin  may  be  separated  from  its  solution  in  chloroform  by  shaking  with 
dilute  caustic  alkali  (differing  from  lutein).  Solutions  of  alkali  bilirubin 
in  water  are  precipitated  by  the  soluble  salts  of  the  alkaline  earths  and 
also  by  metallic  salts. 

As  Ehrlich  first  showed,  bilirubin  forms  combinations  with  diazo 
compounds,  which  have  been  closely  studied  by  Proscher,  Orxdorff  and 
Teeple.i  a  test  suggested  by  Ehrlich  for  bilirubin  is  based  upon  this 
behavior  with  diazobenzenesulphonic  acid. 

If  an  alkaline  solution  of  bilirubin  be  allowed  to  stand  in  contact  with 
the  air,  it  gradually  absorbs  oxygen,  and  green  biliverdin  is  formed.  This 
process  is  accelerated  by  warming.  According  to  Kuster,  in  this  case 
the  alkali  also  has  a  splitting  action  upon  the  pigment,  and  not  one  bodv  but 
several  are  formed.  Biliverdin  is  also  formed  from  bilirubin  by  oxidation 
under  other  conditions.  A  green  coloring-matter  similar  in  appearance  is 
formed  by  the  action  of  other  reagents  such  as  CI,  Br,  and  I.  According  to 
Jolles,^  by  the  use  of  Hijbl's  iodine  solution  biliverdin  is  produced,  while 
according  to  others  (Thudichum.  Maly  ^)  substitution  products  of  bili- 
rubin are  formed. 

Gmelin's  Reaction  for  Bile-pigments.  If  one  carefully  pours  under  an 
aqueous  solution  of  alkali  bilirubin  nitric  acid  containing  some  nitrous  acid, 
there  is  obtained  a  series  of  colored  layers  at  the  juncture  of  the  two  liquids 
in  the  following  order  from  above  downwards:   green,    blue,   \-iolet,   red. 

'  Ehrlich,  Zeitschr.  f.  anal.  Chem.,  23;  Proscher,  Zeitschr.  f.  physiol.  Chem.,  39; 
Omdorff  and  Teeple,  1.  c. 

'  Kiist-er,  Ber.  d.  d.  chem.  Gesellsch.,  35;  JoUes,  Journ.  f.  prakt.  Chem.  (N.  F.),  59, 
and  Pfliiger's  -\rch.,  75. 

'Thudichum,  Joum.  of  Chem.  Soc.  (2),  13,  and  Journ.  f.  prakt.  Chem.  (N.  F.), 
53;   Maly,  Wien.  Sitzimgsber.,  72. 


322  THE  LIVER. 

and  reddish  yellow.  This  color  reaction,  Gmelin's  test,  is  very  delicate 
and  serves  to  detect  the  presence  of  one  part  bilirubin  in  80  000  parts 
liquid.  The  green  ring  must  never  be  absent;  and  also  the  reddish-violet 
must  be  present  at  the  same  time,  otherwise  the  reaction  may  be  confused 
with  that  for  lutein,  which  gives  a  blue  or  greenish  ring.  The  nitric  acid 
must  not  contain  too  much  nitrous  acid,  for  then  the  reaction  takes  place 
too  quickly  and  it  does  not  become  typical.  Alcohol  must  not  be  present 
in  the  liquid,  because,  as  is  well  known,  it  gives  a  play  of  colors,  in  green 
or  blue,  with  the  acid. 

Hammarstex's  Reaction.  An  acid  is  first  prepared  consisting  of  1  vol. 
nitric  acid  and  19  vols,  hydrochloric  acid  (each  acid  being  about  25  per 
cent).  One  volume  of  this  acid  mixture,  which  can  be  kept  for  at  least 
a  year,  is,  when  it  has  become  yellow  by  standing,  mixed  with  4  vols. 
alcohol.  If  a  drop  of  bilirubin  solution  is  added  to  a  few  cubic  centimetres 
of  this  colorless  mixture  a  permanent  beautiful  green  color  is  obtained 
immediately.  On  the  further  addition  of  the  acid  mixture  to  the  green 
liquid  all  the  colors  of  Gmelin's  scale,  as  far  as  choletelin,  can  be  produced 
consecutively. 

Huppert's  Reaction.  If  a  solution  of  alkali  bilirubin  is  treated  with 
milk  of  lime  or  with  calcium  chloride  and  ammonia,  a  precipitate  is  pro- 
duced consisting  of  calcium  bilirubin.  If  this  moist  precipitate,  which  has 
been  washed  with  water,  is  placed  in  a  test-tube  and  the  tube  half  filled 
with  alcohol  which  has  been  acidified  with  hydrochloric  acid,  and  heated 
to  boiling  for  some  time,  the  liquid  becomes  emerald-green  or  bluish  green 
in  color. 

In  regard  to  the  modifications  of  Gmelin's  test  and  certain  other 
reactions  for  bile-pigments,  see  Chapter  XV  (Urine). 

That  the  characteristic  play  of  colors  in  Gmelin's  test  is  the  result  of 
an  oxidation  is  generally  admitted.  The  first  oxidation  step  is  the  green 
biliverdin.  Then  follows  a  blue  coloring-matter  which  Heinsius  and 
Campbell  call  hiliciianin  and  Stokvis  calls  cholecyanin,  and  which  shows  a 
characteristic  absorption-spectrum.  The  neutral  solutions  of  this  color- 
ing-matter are,  according  to  Stokvis,  bluish  green  or  steel-blue  with  a 
beautiful  blue  fluorescence.  The  alkaline  solutions  are  green  and  have 
no  marked  fluorescence,  and  show  three  absorption-bands:  one,  sharp  and 
dark,  in  the  red  between  C  and  D,  nearer  to  C;  a  second,  less  well  defined, 
covering  D;  and  a  third  between  E  and  F,  near  E.  The  strongly  acid 
solutions  are  violet-blue  and  show  two  bands,  described  by  Jaff6,  between 
the  lines  C  and  E,  separated  from  each  other  by  a  narrow  space  near  D.  A 
third  band  between  h  and  F  is  seen  with  difficulty.  The  next  oxidation 
step  after  these  blue  coloring-matters  is  a  red  pigment,  and  lastly  a  yellow- 
ish-brown pigment,  called  choletelin  by  Maly,  which  in  neutral  alcoholic 
solutions  does  not  give  any  absorption-spectrum,  but    in  acid  solution 


BILIVERDIN.  323 

gives  a  band  between  b  and  F.  On  oxidizing  cholecyanin  with  lead  per- 
oxide, Stokvis  1  obtained  a  product  which  he  calls  choletelin,  which  is 
quite  similar  to  urmary  urobilin,  to  be  discussed  later. 

Bilirubin  is  best  prepared  from  gall-stones  of  oxen,  these  concretions 
being  very  rich  in  calcium  bilirubin.  The  finely  powdered  concrement  is 
first  exhausted  with  ether  and  then  with  boiling  water,  so  as  to  remove  the 
cholesterin  and  bile-acids.  In  order  to  remove  the  mineral  constituents 
it  is  better  to  use  10  per  cent  acetic  acid  instead  of  hydrochloric  acid 
(KusTER-).  A  green  pigment  is  now  removed  by  extraction  with  alcohol, 
and  the  choleprasia  is  extracted  with  hot  glacial  acetic  acid.  After 
washing  with  water  it  is  dried,  and  extracted  rej^eatedly  with  boiling 
chloroform.  The  bilirubin  separates  from  the  chloroform  as  crtists.  which 
are  treated  once  or  twice  in  the  above  manner.  It  Ls  then  extracted  ^^■ith 
alcohol  and  precipitated  from  its  chloroform  solution  by  alcohol  or  crys- 
tallized from  dimethylaniline. 

The  chloroform  solution  which  separates  from  the  crusts  of  bilirubin  eon- 
tains,  according  to  Kuster.  a  pigment  related  tobilirtibin.  poorer  in  nitrogen, 
also  precipitable  by  alcohol,  and  verc-  readily  soluble  in  chloroform.  This 
has  been  substantiated  by  Orxdorff  and  Teeple.^-  This  pigment,  according 
to  KfsTER.  is  a  transformation  product  of  bilirubin  which  is  rich  in  chlorine. 

The  quantitative  estimation  of  bilirubin  may  be  made  by  the  spectro- 
photometric  method,  according  to  the  steps  suggested  for  the  blood- 
coloring  matters. 

Biliverdin,  C16H18N2O4  or  C32H36X4OS.  This  body,  which  is  formed 
by  the  oxidation  of  bilirubin,  occurs  in  the  bile  of  many  animals,  in  vomited 
matter,  in  the  placenta  of  the  bitch  (?"),  in  the  shells  of  birds'  eggs,  in  the 
urine  in  icterus,  and  sometimes  in  gall-stones,  although  in  ven,-  small 
quantities. 

Biliverdin  is  amorphous;  at  least  it  has  not  been  obtained  in  well- 
defined  crv'stals.  It  is  insoluble  in  water,  ether,  and  chloroform  (this  is 
true  at  least  for  the  artificially  prepared  biliverdin).  but  is  soluble  in  alcohol 
or  glacial  acetic  acid,  showing  a  beautiful  green  color.  It  is  dissolved  by 
alkalies.  gi\"ing  a  brownish-green  color,  and  this  solution  is  precipitated  by 
acids,  as  well  as  by  calcium,  barium,  and  lead  salts.  Biliverdin  gives 
Huppert's,  Gmelix's,  and  HA^nL^RSTEx's  reactions,  commencing  with 
the  blue  color.  It  is  converted  into  hydrobilirubin  by  nascent  hydrogen. 
On  allowing  the  green  bile  to  stand,  also  by  the  action  of  ammonium  sul- 
phide,   the    biliverdin    may   be    reduced    to    bilirubin    (Haycr.ift    and 

SCOFIELD  4). 

'  Heinsius  and  Campbell,  Pfliiger's  Arch.,  4;  Stok\-is,  Centralbl.  f.  d.  med.  Wis- 
eensch.,  1872,  785;  ibid.,  1873,  211  and  449;  Jaffe,  ibid.,  186S;  Maly,  Wien.  Sitzungs- 
ber.,  59. 

'  Zeitschr.  f.  physici.  Chem..  47. 

'  Kuster,  Ber.  d.  d.  chem.  Gesellsch.,  So;   Omdorff  and  Teeple.  1.  c. 

♦Centralbl.  f  Physiol.,  3,  222,  and  Zeitschr.  f.  physiol.  Chem.,  14. 


324  THE    LIVER. 

Biliverdin  is  most  simply  prepared  by  allowing  a  thin  layer  of  an 
alkaline  solution  of  bilirubin  to  stand  exposed  to  the  air  in  a  dish  until  the 
color  is  brownish  green.  The  solution  is  then  precipitated  by  hydro- 
chloric acid,  the  precipitate  washed  with  water  until  no  HCl  reaction  is 
obtained,  then  dissolved  in  alcohol  and  the  pigment  again  separated  by 
the  addition  of  water.  Any  bilirubin  present  may  be  removed  by  means 
of  chloroform.  Hugounenq  and  Doyon  ^  prepared  biliverdin  from  bili- 
rubin by  the  action  of  sodium  peroxide  and  a  little  acid. 

Choleprasin  is  a  green  pigment  isolated  by  Kijster  ^  from  gall-stones,  which  is 
soluble  in  glacial  acetic  acid  but  insoluble  in  alcohol.  It  differs  from  the  other 
bile-pigments  by  containing  sulphur.  On  distillation  with  zinc  powder  it  gives 
the  pyrrol  reaction,  and  on  oxidation  with  chromic  acid,  Kijster  could  not  ob- 
serve any  formation  of  hsematinic  acid. 

Bilifuscin,  so  named  by  Stadeler,^  is  an  amorphous  brown  pigment  soluble 
in  alcohol  and  alkalies,  nearly  insoluble  in  water  and  ether,  and  soluble  with 
great  difficulty  in  chloroform  (when  bilirubin  is  not  present  at  the  same  time). 
Pure  bilifuscin  does  not  give  Gmelin's  reaction.  This  is  also  true  for  the  bilifuscin 
prepared  by  v.  Zumbusch,^  which  is  more  like  a  humin  substance  and  the  formula 
of  which  is  C64Hgf,N70i4.  Bilifuscin  has  been  found  in  gall-stones.  Biliprasin  is 
a  green  pigment  prepared  by  Stadbler  from  gall-stones,  which  is  generally  con- 
sidered as  a  mixture  of  biliverdin  and  bilirubin.  Dastre  and  Floresco,*  on  the 
contrary,  consider  biliprasin  as  an  intermediate  step  between  bilirubin  and  bili- 
verdin. According  to  them  it  occurs  as  a  physiological  pigment  in  the  bladder- 
bile  of  several  animals  and  is  derived  from  bilirubin  by  oxidation.  This  oxidation 
is  brought  about  by  an  oxidative  ferment  existing  in  the  bile.  Bilihurain  is  the 
name  given  by  Stadeler  to  that  brownish  amorphous  residue  which  is  left  after 
extracting  gall-stones  with  chloroform,  alcohol,  and  ether.  It  does  not  give 
Gmelin's  test.  Bilicyanin  is  also  found  in  human  gall-stones  (Heinsius  and 
Campbell).  Cholohcematin,  so  called  by  MacMunn,  is  a  pigment  often  occurring 
in  sheep-  and  ox-bile  and  characterized  by  four  absorption-bands,  which  is 
formed  from  hsematin  by  the  action  of  sodium  amalgam.  In  the  dried  condition, 
as  when  obtained  by  the  evaporation  of  the  chloroform  solution,  it  is  green,  and  in 
alcoholic  solution  olive-brown.  This  pigment,  which  has  also  been  found  by 
Hammarsten  in  the  bile  from  the  musk-ox  and  hippopotJimus,  is,  according  to 
Marchlewski,  identical  ^vith  the  crystalline  bilipurpurin  isolated  by  Loebisch 
and  Fischler  from  ox-bile.  This  latter  pigment,  according  to  Marchlewski,  is 
not  a  bile-pigment,  but  phylloerythrin,  a  transformation  product  of  chlorophyll. 
Phylloerythrin  has  been  detected  by  Marchlewski  *  in  the  excrement  of  cows 
fed  on  green  grass. 

Gmeijn's  and  Huppert's  reactions  are  generally  used  to  detect  the 
presence  of  bile-pigments  in  animal  fluids  or  tissues.  The  first,  as  a  rule, 
can  be  performed  directly,  and  the  presence  of  proteins  does  not  interfere 
■with  it,  but,  on  the  contrary,  it  brings  out  the  play  of  colors  more  strik- 

'  Arch,  de  Physiol.  (5),  8. 

^  Zeitschr.  f.  physiol.  Chem.,  47. 

^  Cited  from  Hoppe-Seyler,  Physiol,  u.  Path.  chem.  Analyse,  6.  Aufi.,  p.  225. 

*  Zeitschr.  f.  physiol.  Chem.,  31. 
«  Arch,  de  Physiol.  (5),  9. 

•  MacMunn,  Journ.  of  Physiol.,  6;  Loebisch  and  Fischler,  Wien.  Sitzungsber.,  112 
(1903);  Marchlewski,  Zeitschr.  f.  physiol.  Chem.,  41,  43,  and  45;  Hammarsten,  ibid.,  43, 
and  investigations  not  published. 


COMPOSITION  OF  THE  BILE.  325 

ingly.  If  blood-coloring  matters  are  present  at  the  same  time,  the  bile- 
coloring  matters  are  first  precipitated  by  the  addition  of  sodium  phosphate 
and  milk  of  lime.  This  precipitate  containing  the  bile-pigments  may  be 
used  directly  in  Huppert's  reaction,  or  a  little  of  the  precipitate  may  be 
dissolved  in  Hamniarsten's  reagent.  Bilirubin  is  detected  in  blood, 
according  to  Hedexius,^  by  precipitating  the  proteins  with  alcohol,  filtering 
and  acidifying  the  filtrate  with  hydrochloric  or  sulphuric  acid,  and  boiling. 
The  liquid  becomes  of  a  greenish  color.  Serum  and  serous  fluids  may  be 
boiled  directly  with  a  little  acid  after  the  addition  of  alcohol. 

Besides  the  bile-acids  and  the  bile-pigments,  there  occur  in  the  bile  also 
cholcsterin,  lecithin,  jecorin  or  other  phosphatides  (Ham.marstex),  palmitin, 
stearin,  olein,  myristic  acid  (Lassar-Cohx  -),  soaps,  ethereal  sulphuric  acids, 
conjugated  glucuronates,  diastatic  and  proteolytic  enzymes.  Choline  and 
glycerophosphoric  acid,  when  they  are  present,  may  be  considered  as  decom- 
position products  of  lecithin.  Urea  occurs,  though  onh'  in  traces,  as  a 
physiological  constituent  of  human,  ox,  and  dog  bile.  Urea  occurs  in  the 
bile  of  the  shark  and  ray  in  such  large  quantities  that  it  forms  one  of  the 
chief  constituents  of  the  bile.^  The  mineral  constituents  of  the  bile  are, 
besides  the  alkalies,  to  which  the  bile-acids  are  united,  sodium  and  potas- 
sium chloride,  calcium  and  magnesium  phosphate,  and  iron — 0.04-0.115 
p.  m.  in  human  bile,  chiefly  combined  with  phosphoric  acid  (Youxg^). 
Traces  of  copper  are  habitually  present,  and  traces  of  zinc  are  often  found. 
Sulphates  are  entirely  absent,  or  occur  only  in  very  small  amounts. 

The  quantity  of  iron  in  the  bile  varies  greatly.  According  to  Novi  it 
is  dependent  upon  the  kind  of  food,  and  in  dogs  it  is  lowest  with  a  bread 
diet  and  highest  with  a  meat  diet.  According  to  Dastre  this  is  not  the 
case.  The  quantity  of  iron  in  the  bile  varies  even  though  a  constant  diet  is 
maintained,  and  the  variation  is  dependent  upon  the  formation  and  destruc- 
tion of  blood.  According  to  Beccari  ^  the  iron  does  not  disappear  from 
the  bile  in  inanition,  and  the  percentage  shows  no  constant  diminution. 
The  question  as  to  the  extent  of  elimination  by  the  bile  of  the  iron  intro- 
duced into  the  body  has  received  various  answers.  There  is  no  doubt 
that  the  liver  has  the  property  of  collecting  and  retaining  iron  as  well  as 
other  metals  from  the  blood.  Certain  investigators,  such  as  Xo\t  and 
KuxKEL,  are  of  the  opinion  that  the  iron  introduced  and  transitorily 
retained  in  the  liver  is  eliminated  by  the  bile,  while  others,   such  as  Ham- 


'  Upsala  Lakaref.  Forh.,  29,  and  Maly's  Jahresber.,  24. 
'  Zeitschr.  f.  physiol.  Chem.,  17;  Hammersten,  ibid.,  32,  36  and  43. 
'  Hammarsten,  ibid.,  24. 
*  Journ.  of  Anat.  and  Physiol.,  5.  158. 

^  Novi,  see  Maly's  Jahresber.,  20;    Dastre,  Arch,  de  Physiol.  (5),  3;  Beccari,  Arch, 
ital.  de  Biol.,  28. 


326  THE  LIVER. 

BURGER,  Gottlieb,  and  Anselm,^  deny  any  such  elimination  of  iron  by 
the  bile. 

Quantitative  Composition  of  the  Bile.  Complete  analyses  of  human  bile 
have  been  made  by  Hoppe-Seyler  and  his  pupils.  The  bile  was  removed 
as  fresh  as  possible  from  the  gall-bladder  of  those  cadavers  the  livers  of 
which  were  in  no  sense  pathological. 

Older  and  less  complete  analyses  of  human  bile  have  been  made  by 
Frerichs  and  v.  Gorup-Besanez.^  The  bile  analyzed  by  them  was  from 
perfectly  healthy  persons  who  had  been  executed  or  accidentally  killed. 
The  two  analyses  of  Frerichs  are,  respectively,  of  (I)  an  IS-year-old  and 
(II)  a  22-year-old  male.  The  analyses  of  v.  Gorup-Besanez  are  of  (I)  a 
man  of  49  and  (II)  a  woman  of  29.  The  results  are,  as  usual,  in  parts 
per  1000. 

Frerichs.  v.  Gorup-Besanez. 

I.  II.  I.  II. 

Water 860.0  859.2  822.7  898.1 

Solids 140.0  140.8  177.3  101.9 

Biliary  salts 72.2  91.4  107.9  56.5 

Mucus  and  pigments 26 . 6  29 . 8  22 . 1  14.5 

Cholesterin 1.6  2.61  .-q  ono 

Fat 3.2  9.2/  ^^'^  ""^'^ 

Inorganic  substances  .  .  ..  6.5  7.7  10,8  6.2 

Human  liver-bile  is  poorer  in  solids  than  the  bladder-bile.  In  several 
cases  it  contained  only  12-18  p.  m.  solids,  but  the  bile  in  these  cases  is 
hardly  to  be  considered  as  normal.  Jacobsen  found  22.4^22.8  p.  m.  solids 
in  a  specimen  of  bile.  Hamalirsten,  who  had  occasion  to  analj-ze  the 
liver-bile  in  seven  cases  of  biliar}-  fistula,  has  repeatedly  found  25-28  p.  m. 
solids.  In  a  case  of  a  corpulent  woman  the  quantity  of  solids  in  the  liver- 
bile  varied  between  .30.10-38.6  p.  m.  in  ten  days.  Brand  3  has  observed 
still  higher  figures,  more  than  40  p.  m.  in  a  couple  of  cases.  This  investi- 
gator suggests  that  the  bile  from  an  imperfect  fistula,  when  it  is  partly 
absorbed,  is  richer  in  solids  than  when  it  comes  from  a  perfect  fistula. 

The  molecular  concentration  of  human  bile,  according  to  Brand, 
BoNANNi,  and  Strauss,^  is  nearly  always  identical  with  that  of  the  blood, 
although  the  amount  of  water  and  solids  varies.  The  freezing-point  varies 
only  between  —0.54°  and  —0.58°.     This  constancy  of  the  osmotic  pressure 

'  Kunkel,  Pfliiger's  Arch.,  14;  Hamburger,  Zeitschr.  f.  piiysiol.  Chem.,  2  and  4; 
Gottlieb,  zbid.,  15;  Anselm,  ''Ueber  die  Eisenausscheidung  der  Galle,"  Inaug.-Diss. 
Dorpat,  1891.     See  also  the  works  cited  in  foot-note  3,  p.  244. 

=  See  Hoppe-Seyler,  Physiol.  Chem.,  301;  Socoloff,  Pfliiger's  Arch.,  12;  Trifanow- 
ski,  ibid.,  9;   Frerichs  m  Hoppe-Seyler 's  Physiol.  Chem.,  299;   v.  Gorup-Besanez,  ibid. 

*  Jacobsen,  Ber.  d.  deutsch.  chem.  Gesellsch.,  6;  Hammarsten,  Nova  Acta  Reg. 
Soc   Scient.  Upsala,  16;    Brand.  Pfliiger's  Arch.   90. 

'Brand,  1.  c  ;  Bonanni.  Biochem.  Centralbl.,  1;  Strauss,  Berl.  klin.  Wochenschr., 
1903. 


COMPOSITION  OF  THE   BILE.  327 

is  explained  by  the  fact  that  in  concentrated  biles  with  larger  amounts  of 
organic  substances  (with  larger  molecules)  the  amount  of  inorganic  salts 
is  lower.i 

Human  bile  sometimes,  but  not  always,  contains  sulphur  in  an  ethereal 
sulphuric-acid-like  combination  (Hammarsten,  Oerum,  Brand).  The 
quantity  of  such  sulphur  may  even  amount  to  J-"^  of  the  total  sulphur. 
We  do  not  know  the  nature  of  these  ethereal  sulphuric  acids.  According 
to  Oerum  ^  they  are  not  precipitated  by  lead  acetate,  but  are  precipitated 
by  basic  lead  acetate,  especially  with  ammonia.  Human  bile  is  habitually 
richer  in  glycocholic  than  in  taurocholic  acid.  In  six  cases  of  liver-bile 
analyzed  by  Hammarsten  the  relationship  of  taurocholic  to  glycocholic 
acid  varied  between  1:2.07  and  1:14.36.  The  bile  analyzed  by  Jacobsen 
contained  no  taurocholic  acid. 

As  an  example  of  the  composition  of  human  liver-bile  the  following 
results  of  three  analyses  made  by  Hammarstex  are  given.  The  results 
are  calculated  in  parts  per  1000.3 

Solids 25.200  35.260  25.400 

Water 974.800  964.740  974.600 

Mucin  and  pigments 5 .  290  4 .  290  5 .  150 

Bile-salts 9.310  18.240  9.040 

Taurocholate 3 .  034  2 .  079  2 .  180 

Glycocholate 6.276  16.161  6.860 

Fatty  acids  from  soaps 1 .  230  1 .  360  1.010 

Cholesterin 0.630  1.600  1 .500 

Lecithin 1     ^  r,.-,^  0 .  574  0 .  650 

Fat /      -^  -^  0.956  0.610 

Soluble  salts 8.070  6.760  7.250 

Insoluble  salts .       0.250  0.490  0.210 

Among  the  mineral  constituents  the  chlorine  and  sodium  occur  to  the 
greatest  extent.  The  relationship  between  potassium  and  sodium  varies 
considerably  in  different  samples.  Sulphuric  acid  and  phosphoric  acid 
occur  only  in  very  small  quantities. 

Bagixsky  and  Sommerfeld  "*  have  found  true  mucin,  mixed  with 
some  nucleoalbumin,  in  the  bladder-bile  of  children.  The  bile  contained 
on  an  average  896.5  p.  m.  water;  103.5  p.  m.  solids;  20  p.  m.  mucin;  9.1 
p.  m.  mineral  substances;  25.2  p.  m.  bile-salts  (of  which  16.3  p.  m.  were 
glycocholate  and  8.9  p.  m.  taurocholate);  3.4  p.  m.  cholesterin;  6.7  p.  m. 
fat,  and  2.8  p.  m.  leucine.^ 

The  quantity  of  pigment  in  human  bile  is,  according  to  Noel-Paton, 
0.4-1.3  p.  m.  (in  a  case  of  biliary  fistula).     The  method  used  in  determining 

'  See  Brand,  1.  c;   Hammarsten,  1.  c. 

2  Skand.  Archiv  f.  Physiol.,  16. 

3  Recent  quantitative  analyses  may  be  found  in  Brand,  1.  c;  v.  Zeynek,  Wien, 
klin.  Wochenschr.,  1899;   Bonanni,  1.  c. 

^  Verhandl.  d.  physiol.  Gesellsch.  zu  Berlin,  1894-95. 

*  Analyses  of  bile  from  children  may  be  found  in  Heptner,  Maly's  Jahresber.,  30. 


328  THE  LIVER. 

the  pigments  in  this  case  was  not  quite  trustworthy.  More  exact  results 
obtained  by  spectrophotometric  methods  are  on  record  for  dog-bile. 
According  to  Stadelmaxx  ^  dog-bile  contains  on  an  average  0.6-0.7  p.  m. 
bilirubin.  At  the  most  only  7  milligrams  of  pigment  are  secreted  per  kilo 
of  body  in  the  twenty-four  hours. 

In  animals  the  relative  proportion  of  the  two  acids  varies  considerably. 
It  has  been  found,  on  determining  the  amount  of  sulphur,  that,  so  far  as 
the  experiments  have  gone,  taurocholic  acid  is  the  prevailing  acid  in  car- 
nivorous mammals,  birds,  snakes,  and  fishes.  Among  the  herbivora, 
sheep  and  goats  have  a  predominance  of  taurocholic  acid  in  the  bile.  Ox- 
bile  sometimes  contains  taurocholic  acid  in  excess,  in  other  cases  glyco- 
cholic  acid  predominates,  and  in  a  few  cases  the  latter  occurs  almost  alone. 
The  bile  of  the  rabbit,  hare,  kangaroo,  hippopotamus,  and  orang-utang 
(Hammarsten^)  contains,  like  the  bile  of  the  pig,  almost  exclusively  glyco- 
eholic  acid.  A  distinct  influence  on  the  relative  amounts  of  the  two  bile- 
acids  exerted  by  differences  in  diet  has  not  been  detected.  Ritter  ^  claims 
to  have  found  a  decrease  in  the  quantity  of  taurocholic  acid  in  calves  when 
they  pass  from  the  milk  to  the  vegetable  diet. 

In  the  above-mentioned  calculation  of  the  taurocholic  acid  from  the 
quantity  of  sulphur  in  the  bile-salts  it  must  be  remarked  that  no  exact  con- 
clusion can  be  drawn  from  such  a  determination,  since  it  is  known  that 
other  kinds  of  bile  (e.g.,  human  and  shark  bile)  contain  sulphur  in  com- 
pounds other  than  taurocholic  acid.* 

The  phosphorized  constituents  of  bile  are  not  well  known;  nevertheless^ 
there  is  no  doubt  that  bile  contains  other  phosphatides  besides  lecithin 
(Hammarstex).  These  phosphatides  are  in  part  precipitated  in  the 
precipitation  of  the  bile-salts  and  they  in  part  keep  the  bile-salts  in  solution, 
preventing  their  complete  precipitation,  and  hence  they  have  a  double 
disturbing  action  in  the  quantitative  analysis  of  bile.  Those  biles  richest 
in  phosphatides,  so  far  as  known,  are  the  following,  in  the  order  of 
their  amount:  polar  bear,  man  (in  special  cases),  dog,  black  bear,  orang- 
utang.  The  bile  of  certain  fishes  contains  but  little  phosphatides  (Ham- 
marsten^). 

The  cholesterin,  which,  according  to  several  investigators,  not  only  origi- 
nates from  the  liver,  but  also  from  the  biliary  passages,  occurs  in  larger 
quantities  in  the  bladder-l)ile  than  in  the  liver-bile,  and  is  present  to  a 


'  Noel-Paton,  Rep.  Lab.  Roy.  Soc.  Coll.  Phys.  Edinburgh,    3;    Stadelmann,  Der 
Icterus. 

^  Investigations  not  published.     See  Ergebnisse  der  Physiol.,  4. 

'  Cited  from  Maly's  .Jahresber.,  6,  195. 

*  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  32,  and  Ergebnisse  der  Physiol.,  4. 

'  Zeitschr,  f.  physiol.  Chera.,  36,  and  Ergebnisse  der  Physiol.,  4. 


CHEMICAL   FORMATION    OF    THE   BILE.  329 

greater  extent  in  the  non-filtered  than  in  the  filtered  bile   (Doyon  and 
DUFOURT  '). 

The  gases  of  the  bile  consist  of  a  large  quantity  of  carbon  dioxide,  which 
increases  with  the  amount  of  alkalies,  only  traces  of  oxygen,  and  a  very 
small  quantity  of  nitrogen. 

Little  is  known  in  regard  to  the  properties  of  the  bile  in  disease.  The  quantity 
of  urea  is  found  to  be  considerably  increased  in  uraemia.  Leucine  and  tyrosine  are 
observed  in  acute  yellow  atrophy  of  the  liver  and  in  typhoid.  Traces  of  albumin 
(without  regard  to  nucleoalbumin)  have  several  times  been  found  in  the  human 
bile.  The  so-called  pigmentary  acholia,  or  the  secretion  of  a  bile  containing 
bile-acids  but  no  bile-pigments,  has  also  been  repeatedly  noticed.  In  all  such 
cases  observed  by  Rittee  he  found  a  fatty  degeneration  of  the  liver-cells,  in  re- 
turn for  which,  even  in  excessive  fatty  infiltration,  a  normal  bile  containing  pig- 
ments was  secreted.  The  secretion  of  a  bile  nearly  free  from  bile-acids  has  been 
observed  by  Hoppe-Seyler  '  in  amyloid  degeneration  of  the  liver.  In  animals, 
dogs,  and  especially  rabbits  it  has  been  observed  that  the  blood-pigments  pass 
into  the  bile  in  poisoning  and  in  other  conditions,  causing  a  destruction  of  the 
blood-corpuscles,  as  also  after  intravenous  haemoglobin  injection  (Wertheimer 
and  Meyer,  Filehne,  Stern  ^).  Albumin  can  pass  into  the  bile  after  the  intra- 
venous injection  of  a  foreign  protein  (casein)  (GtfRBER  and  Hallauer),  as  well 
as  after  poisoning  with  phosphorus  or  arsenic  (Pilzecker),  or  after  the  irrita- 
tion of  the  liver  by  the  introduction  of  ethyl  alcohol  or  amyl  alcohol  (Brauer). 
Sugar  occurs  in  bile  only  in  exceptional  cases.* 

The  physiological  secretion  of  the  gall-bladder  is  according  to  Wahl- 
gren^  in  man  a  viscous,  alkaline  fluid  with  11.24-19.63  p.  m.  solids  The 
mucilaginous  properties  are  not  due  to  mucin  but  to  a  phosphorized  pro- 
tein substance  (nucleoalbumin  or  nucleoproteid). 

Instead  of  bile  there  is  sometimes  found  in  the  gall-bladder  under  pathological 
conditions  a  more  or  less  viscous,  thready,  colorless  fluid  which  contains  pseudo- 
mucins  or  other  peculiar  protein  substances.^ 

Chemical  Formation  of  the  Bile.  The  first  question  to  be  answered  is 
the  following:  Do  the  specific  constituents  of  the  bile,  the  bile-acids  and 
bile-pigments,  originate  in  the  liver;  and  if  this  is  the  case,  do  they  come 
from  this  organ  alone,  or  are  they  also  formed  elsewhere? 

The  investigations  of  the  blood,  and  especially  the  comparative  investi- 
gations of  the  blood  of  the  portal  and  hepatic  veins  under  normal  condi- 
tions, have  not  given  any  answer  to  this  question.     To  decide  this,  therefore, 

*  Arch,  de  Physiol.  (5),  8. 

^  Ritter,  Compt.  rend.,  74,  and  Journ.  de  I'anat.  et  de  la  physiol.  (Robin),  1872; 
Hoppe-Seyler,  Physiol.  Chem.,  317. 

'  Wertheimer  and  Meyer,  Compt.  rend.,  108;  Filehne,  Virchow's  Arch.,  121;  Stern, 
ibid.,  123. 

*  Giirber  and  Hallauer,  Zeitschr.  f.  Biologie,  45;  Pilzecker,  Zeitschr.  f.  physioL 
Chem.,  41;  Brauer,  ibid.,  40. 

'  See  Maly's  Jahresber.,  Z2. 

'Winternitz,  Zeitschr.  f.  physiol.  Chem.,  21;    SoUmann,  Amer.  Medicine,  5  (1903) 


330  THE   LIVER. 

it  is  necessary  to  extirpate  the  liver  of  animals  or  to  isolate  it  from  the 
circulation.  If  the  bile  constituents  are  not  formed  in  the  liver,  or  at  least 
not  alone  in  this  organ,  but  are  eliminated  only  from  the  blood,  then,  after 
the  extirpation  or  removal  of  the  liver  from  the  circulation,  a:i  accumulation 
of  the  bile  constituents  is  to  be  expected  in  the  blood  and  tissues.  If  the 
bile  constituents,  on  the  contrary,  are  formed  exclusively  in  the  liver,  then 
the  above  operation  naturally  would  give  no  such  result.  If  the  ductus 
choledochus  is  tied,  then  the  bile  constituents  will  be  collected  in  the  blood 
or  tissues  whether  they  are  formed  in  the  liver  or  elsewhere. 

From  these  principles  Kobner  has  tried  to  demonstrate  by  experiments 
on  frogs  that  the  bile-acids  are  produced  exclusively  in  the  liver.  While  he 
was  unable  to  detect  any  bile-acids  in  the  blood  and  tissues  of  these  animals 
afte  •  extirpation  of  the  liver,  he  was  able  to  discover  them  on  tying  the 
ductus  choledochus.  The  investigations  of  Ludwig  and  I  leischl  i  show 
that  in  t'  e  dog  the  bile-acids  originate  in  the  liver  alone.  After  tying  the 
ductus  choledochus  they  observed  that  the  bile  constituents  were  absorbed 
by  the  lymphatic  vessels  of  the  liver  and  passed  into  the  blood  through  the 
thoracic  duct.  Bile-acids  could  be  detected  in  the  blood  after  such  an 
operation,  while  they  could  not  be  detected  in  the  normal  blood.  But 
Avhen  the  common  bile  and  thoracic  ducts  were  both  tied  at  the  same  time, 
then  not  the  least  trace  of  bile-acids  could  be  detected  in  the  blood,  while 
if  they  are  also  formed  in  other  organs  and  tissues  they  should  have  been 
present. 

From  older  statements  of  Cloez  and  Vulpian,  as  well  as  Virchow,  the  bile- 
acids  also  occur  in  the  suprarenal  capsule.  These  statements  have  not  been 
confirmed  by  later  investigations  of  Stadelmann  and  Beier.^  At  the  present 
time  there  is  no  ground  for  supposing  that  the  bile-acids  are  formed  elsewhere 
than  in  the  liver. 

It  has  been  indubitably  proved  that  the  bile-pigments  may  be  formed  in 
other  organs  besides. the  liver,  for,  as  is  generally  admitted,  the  coloring- 
matter  hsematoidin,  which  occurs  in  old  blood  extravasations,  is  identical 
with  the  bile-pigment  bilirubin  (see  page  320).  LATsckENBERGER^  has 
also  observed  in  horses,  under  pathological  conditions,  a  formation  of  bile- 
pigments  from  the  blood-coloring  matters  in  the  tissues.  Also  the  occur- 
rence of  bile-pigments  in  the  placenta  seems  to  depend  on  their  formation 
in  that  organ,  while  the  occurrence  of  small  quantities  of  bile-pigments  in 
the  blood-serum  of  certain  animals  probably  depends  on  an  absorption  of 
these  substances. 


*  Kobner,  see  Heidenhain,  Physiologie  tier  Absonderungsvorgange,  in  Hermann's 
Handbuch,  5;  FleiscU,  Arbeiten  aus  der  physiol.  Anstalt  zu  Leipzig,  Jahrgang  9. 
'  Zeitschr.  f.  physiol.  Chem.,  IS,  in  which  the  older  literature  may  be  found. 
^  See  Maly's  Jahresber.,  16,  and  Monatshefte  f.  Chem.,  9. 


CHEMICAL    FORMATION   OF  THE    BILE.  331 

Although  the  bile-pigments  may  be  formed  in  other  organs  besides  the 
liver^  stin  it  is  of  first  importance  to  know  what  bearing  this  organ  has  on 
the  elimination  and  formation  of  bile-pigments.  In  this  regard  it  must  be 
recalled  that  the  liver  is  an  excretory  organ  for  the  bile-pigments  circulat- 
ing in  the  blood.  Tarchanoff  has  observed,  in  a  dog  with  biliary  fistula, 
that  intravenous  injection  of  bilirubin  causes  a  very  considerable  increase  in 
the  bile-pigments  eliminated.  This  statement  has  been  confirmed  lately 
by  the  investigations  of  Vossius.^ 

Numerous  experiments  have  been  made  to  decide  the  question  whether 
the  bile-pigments  are  only  eliminated  by  the  liver  or  whether  they  are  also 
formed  therein.  By  experimenting  on  pigeons,  Stern  was  able  to  detect 
bile-pigments  in  the  blood-serum  five  hours  after  tying  the  biliary  passages 
alone,  while  after  tying  all  the  vessels  of  the  liver  and  also  the  biliary  pas- 
sages, no  bile-pigments  could  be  detected  either  in  the  blood  or  the  tissues 
of  the  animal,  which  was  killed  10-24  hours  after  the  operation.  Min- 
kowski and  Naunyn^  have  also  found  that  poisoning  with  arseniuretted 
hydrogen  produces  a  liberal  formation  of  bile-pigments  and  the  secretion, 
after  a  short  time,  of  a  urine  rich  in  biliverdin  in  previously  healthy  geese. 
In  geese  with  extirpated  livers  this  does  not  occur. 

No  such  experiments  can  be  carried  out  on  mammalia,  as  they  do  not 
live  long  enough  after  the  operation;  still  there  is  no  doubt  that  this  organ 
is  the  chief  seat  of  the  formation  of  bile-pigments  under  physiological  con- 
ditions. 

In  regard  to  the  materials  from  which  the  bile-acids  are  produced,  it 
may  be  said  with  certainty  that  the  two  components,  glycocoU  and  taurine, 
which  are  both  nitrogenized,  are  formed  from  the  protein  bodies.  The 
close  relationship  of  taurine  to  the  cystine  group  of  the  protein  molecule 
has  been  especially  shown  by  the  investigations  of  Friedmann  (see  Chapter 
II),  and  very  recently  v.  Bergmann  ^  has  shoT\Ti  by  feeding  do^s  with  sodium 
cholate  and  cystine  that  the  animal  body  can  transform  cystine  into  taurine 
and  that  the  taurine  of  the  bile  originates  from  the  proteins  of  the  food.  In 
regard  to  the  origin  of  the  non-nitrogenized  cholic  acid,  which  was  formerly 
considered  as  originating  from  the  fats,  nothing  is  known  positively. 

The  blood-coloring  matters  are  considered  as  the  mother-substances  of 
the  bile-pigments.  If  the  identity  of  haematoidin  and  bilirubin  was  settled 
beyond  a  doubt,  then  this  view  might  be  considered  as  proved.  Independ- 
ently, however,  of  this  identity,  which  is  not  admitted  by  all  investigators, 
the  view  that  the  bile-pigments  are  derived  from  th^  blood-coloring  matters 
has  strong  arguments  in  its  favor.     It  has  been  shown  by  several  experi- 

•  Tarchanoff,  Pfliiger's  Arch.,  9;   Vogsius,  cited  from  Stadelmann,  Der  Icterus. 

^  Stern,  Arch.  f.  exp.  Path.  u.  Pharm.,  IJ);   Minkowski  and  Naunyn,  ibid.,  21. 

^  Hofmeister's  Beitrage,  4.     See  also  Wolilgemuth,  Zeitsclir.  f.  physiol.  Chem.,  40o 


332  THE  LIVER. 

menters  that  a  yellow  or  yellowish-red  pigment  can  be  formed  from  the 
blood-coloring  matters,  which  gives  Gmelin's  test,  and  which,  though  it 
may  not  form  a  complete  bile-pigment,  is  at  least  a  step  in  its  formation 
(Latschenbekger).  a  further  proof  of  the  formation  of  the  bile-pigments 
from  the  blood-coloring  matters  consists  in  the  fact  that  hsematin  on  reduc- 
tion yields  urobilin,  which  is  identical  with  hydrobilirubin  (see  Chapter 
XV).  Further,  hsematoporphyrin  (see  page  212)  and  bilirubin  are  isomers, 
according  to  Nencki  and  Sieber,  and  closely  allied.  The  formation  of 
bilirubin  from  the  blood-coloring  matters  is  shown,  according  to  the  obser- 
vations of  several  investigators,^  by  the  appearance  of  free  haemoglobin  in 
the  plasma — produced  by  the  destruction  of  the  red  corpuscles  by  widely 
differing  influences  (see  below)  or  by  the  injection  of  hgemoglobin  solution, 
causing  an  increased  formation  of  bile-pigments.  The  amount  of  pig- 
ments in  the  bile  is  not  only  considerably  increased,  but  the  bile-pigments 
may  even  pass  into  the  urine  under  certain  circumstances  (icterus).  After 
the  injection  of  haemoglobin  solution  into  a  dog  either  subcutaneously  or  in 
the  peritoneal  cavity,  Stadelmann  and  Gorodecki  ^  observed  in  the  secre- 
tion of  pigments  by  the  bile  an  increase  of  61  per  cent,  which  lasted  for 
more  than  twenty-four  hours. 

If  bilirubin,  which  contains  no  iron,  is  derived  from  haematin,  which  con- 
tains iron,  then  iron  must  be  split  off.  This  process  may  be  represented  by  the 
following  formula,  C32H34N405Fe  +  H20-Fe  =  C32H36N406.  The  question 
in  what  form  or  combination  the  iron  is  split  off  is  of  special  interest,  and 
also  whether  it  is  eliminated  by  the  bile.  This  latter  does  not  seem  to  be 
the  case,  at  least  to  any  great  extent.  In  100  parts  of  bilirubin  which  are 
eliminated  by  the  bile  there  are  only  1.4-1.5  parts  iron,  according  to  Kun- 
kel;  while  100  parts  haematin  contain  about  9  parts  iron.  Minkowski 
and  BaserIxN  ^  have  also  found  that  the  abundant  formation  of  bile-pigments 
occurring  in  poisoning  by  arseniuretted  hydrogen  does  not  increase  the 
quantity  of  iron  in  the  bile.  The  quantity  apparently  does  not  seem  to 
correspond  with  that  in  the  decomposed  blood-coloring  matters.  It  follows 
from  the  researches  of  several  investigators  ^  that  the  iron  is,  at  least  chiefly, 
retained  by  the  liver  as  a  ferruginous  pigment  or  protein  substance. 

What  relationship  does  the  formation  of  bile-acids  bear  to  the  forma- 
tion of  bile-pigments?  Are  these  two  chief  constituents  of  the  ])ile  derived 
simultaneously  from  the  same  material,  and  can  we  detect  a  certain  connec- 


'  See  Stadelmann,  Der  Icterus,  etc.   Stuttgart,  1891. 

•"  Sec  Stade  mann    ibid. 

'  Kunkel,  Pfluger's  Arch.,  14;  Minkowski  and  Baserin,  Arch.  f.  exp.  Path.  u. 
Pharm.,  23. 

*  See  Naunyn  and  Minkowski,  Arch.  f.  exp.  Path.  u.  Pliarm.,  21;  Latschenberger, 
1.  c.;   Neu-Tiann,  Virchow's  Arch.   Ill,  and  the  hterature  in  foot-note  2   p    2  2. 


BILE  CONCRETIONS.  333 

tion  between  the  formation  of  biliruljiu  and  bile-acids  in  the  liver?  The 
investigations  of  Stadelmann  teach  us  that  this  is  not  the  case.  With 
increased  formation  of  bile-pigments  the  amount  of  bile-acids  is  decreased, 
and  the  introduction  of  haemoglobin  into  the  liver  strongly  increases  the 
formation  of  bilirubin,  but  simultaneously  strongly  decreases  the  produc- 
tion of  bile-acids.  According  to  Stadelmann  the  formation  of  bile- 
pigments  and  bile-acids  is  duo  to  a  special  activity  of  the  cells. 

An  absorption  of  bile  from  the  liver  and  the  passage  of  the  bile  con- 
stituents into  the  Vjlood  and  urine  occurs  in  retarded  discharge  of  the  bile, 
and  usually  in  different  forms  of  hepatogenic  icterus.  But  bile-pigments 
may  also  pass  into  the  urine  under  other  circumstances,  especially  when 
in  animals  a  solution  or  destruction  of  the  red  blood-corpuscles  takes 
place  through  injection  of  water  or  a  solution  of  biliary  salts,  through 
poisoning  by  ether,  chloroform,  arseniuretted  hydrogen,  phosphorus,  or 
toluylenediamine.  and  in  other  cases.  This  occurs  also  in  man  in  severe 
infectious  diseases.  It  has  also  been  claimed  many  times  that  a  transfor- 
mation of  blood-pigments  into  bile-pigments  occurs  elsewhere  than  in  the 
liver,  namely,  in  the  blood.  Such  a  belief  has  been  made  very  improb- 
able by  the  important  researches  of  Minkowski  and  Naunyn,  Afanassiew, 
SiLBER\L^NN,  and  especially  of  Stadelmann, ^  and  in  some  of  the  above- 
mentioned  cases,  as  after  poisoning  with  phosphorus,  toluylenediamine,  and 
arseniuretted  hydrogen,  it  has  been  disproved  by  direct  experiment. 

The  icterus  is  also  in  these  cases  hepatogenic ;  it  depends  upon  an  absorp- 
tion of  bile-pigments  from  the  liver,  and  this  absorption  seems  to  originate 
in  various  cases  in  somewhat  different  ways.  Thus  the  bile  may  be 
viscous  and  cause  a  congestion  of  bile  by  counteracting  the  low  secretion 
pressure.  In  other  cases  the  fine  biliary  passages  may  be  compressed  by 
an  abnormal  swelling  of  the  liver-cells,  or  a  catarrh  of  the  bile -passages 
may  occur,  causing  a  congestion  of  the  bile  (Stadelmann). 

Bile  Concretions. 

The  concrements  which  occur  in  the  gall-bladder  vary  considerably  in 
size,  form,  and  number,  and  are  of  three  kinds,  depending  upon  the  kind 
and  nature  of  the  bodies  forming  their  chief  mass.  One  group  of  gall- 
stones contains  lime-pigment  as  chief  constituent,  another  cholesterin, 
and  the  third  calcium  carbonate  and  phosphate.  The  concrements  of  the 
last-mentioned  group  occur  very  seldom  in  man.  The  so-called  cholesterin- 
stones  are  those  which  occur  most  frequently  in  man,  while  the  lime-pig- 
ment stones  are  not  found  very  often  in  man,  but  often  in  oxen. 

'  The  literature  belonging  to  this  subject  is  found  in  Stadelmann,  Der  Icterus,  etc., 
Stuttgart,  1891. 


334  THE   LIVER. 

The  pigment-stones  are  generally  not  large  in  man,  but  in  oxen  and 
pigs  they  are  sometimes  found  the  size  of  a  walnut  or  even  larger.  In 
most  cases  the}-  consist  chiefly  of  calcium  bilirubin  with  little  or  no  bili- 
verdin.  Sometimes  also  small  black  or  greenish-black,  metallic-looking 
stones  are  found,  which  consist  chiefly  of  bilifuscin  along  with  biliverdin. 
Iron  and  copper  seem  to  be  regular  constituents  of  pigment-stones.  Man- 
ganese and  zinc  have  also  been  found  in  a  few  cases.  The  pigment-stones 
are  generally  heavier  than  water. 

The  choksterin-stones,  whose  size,  form,  color,  and  structure  may  vary 
greatly,  are  often  lighter  than  w^ater.  The  fractured  surface  is  radiated, 
crystalline,  and  frequently  shows  crystalline,  concentric  layers.  The 
cleavage  fracture  is  waxy  in  appearance,  and  the  fractured  surface  when 
rubbed  by  the  finger-nail  also  becomes  like  wax.  By  rubbing  against  each 
other  in  the  gall-bladder  they  often  become  faceted  or  take  other  remarkable 
shapes.  Their  surface  is  sometimes  nearly  white  and  waxlike,  but  generally 
their  color  is  variable.  They  are  sometimes  smooth,  in  other  cases  they 
are  rough  or  uneven.  The  quantity  of  cholesterin  in  the  stones  varies  from 
642  to  9S1  p.  m.  (Ritter  ^).  The  cholesterin-stones  also  sometimes  contain 
variable  amomits  of  lime-pigments,  which  may  give  them  a  very  changeable 
appearance. 

Cholesterin,  C27H46O  (Obermuller),  or,  as  ordinarily  given,  C27H44O 
(Mauthner  and  Sum  a).  By  the  action  of  concentrated  sulphuric  acid 
or  phosphoric  acid,  and  also  in  other  wa3's,  hydrocarbons  are  obtained, 
which  are  called  cholesteriline,  cholesterone,  and  cholesterilene.  Mauthxer 
and  SuiDA,2  -^y^q  have  closely  studied  these  hydrocarbons,  have  been  able 
to  prepare  a  crystalline  cholesteriline  by  heating  cholesterin  with  anhydrous 
copper  sulphate.  The  hydrocarbons  stand,  according  to  Weyl,^  in  close 
relationship  to  the  terpene  group,  and  the  color  reactions  of  cholesterin 
as  well  as  the  recent  investigations  on  the  constitution  of  this  body  seem 
to  substantiate  this  view^  Very  painstaking  and  thorough  investigations 
on  the  constitution  of  cholesterin  have  been  made,  of  which  we  must 
especially  mention  those  of  Mauthner  and  Suida,  Windaus  and  Stein, 
DiELs  and  Abderhalden.*  Although  these  researches  have  not  lead  to 
positive  conclusions,  still  we  are  justified  in  concluding  that  cholesterin  prob- 
ably consists  of  a  complex  of  five  hydrogenized  rings,  of  which  one  con- 

>  Journ.  de  I'anat.  et  de  la  physiol.  (Robin),  1872. 

^  Obermuller,  Arch.  f.  (Anat.  u.)Fhysiol.,  1889,  and  Zeitschr.  f.  physiol.  Chem.,  15; 
Mauthner  and  Suida,  Wien.  Sitzungsber.,  Math.  Nat.  Klasse,  103,  Abt.  26,  which  also 
contains  the  older  literature. 

'  Arch.  f.  (Anat.  u.)  Physiol,  1886,  p.  182. 

*  Mauthner  and  Suida,  Monatshefte  f.  Chem.,  15,  17,  24;  Windaus,  "tjber  Choles- 
terin." Hab.-Schrift,  Freiburg  i.  B.,  1903,  Ber.  d.  d.  chem.  Geselkch.,  36,  37,  and 
39;  with  Stein,  ibid.,  37;  Diels  and  Abderhalden,  ibid.,  36,  37,  and  39;  G.  Stein, 
"tJber  Cholesterin,"  Inaug.-Dissert.  Freiburg  i.  B.,  1905. 


CHOLESTERIN.  335 

tains  a  double  bondage  and  another  a  secondary  alcohol  group.  Several 
facts  seem  to  make  it  probable  that  cholesterin  stands  close  to  the 
hydrogenized  retene  and  hence  is  a  complicated  terpene.  From  this 
standpoint,  the  close  relationship  between  cholesterin  and  cholic  acid  is  of 
great  interest. 

On  reduction  of  cholesterin,  and  also  of  the  ketone,  cliolesterone,  corre- 
sponding to  cholesterin,  by  means  of  metallic  sodium  in  amyl-alcohol  solu- 
tion, DiELS  and  Abderhalden  obtained  two  isomeric  dihydrocholes- 
tcrincs,  C27H48O,  a-  and  ^-cholestanol,  of  which  the  first  seems  to  be  identical 
with  he  dihydrocholesterin  obtained  by  Neuberg  and  Rauchwerger  1 
by  the  action  of  sodium  in  amyl-alcohol  solution.  The  identity  of  a-choles- 
tanol  with  koprosterin,  which  will  be  mentioned  below,  is  considered  by 
Neuberg  as  not  improbable,  while  Diels  and  Abderhalden  deny  this. 

Cholesterin  occurs  in  small  amounts  in  nearly  all  animal  fluids  and 
juices.  It  occurs  only  rarely  in  the  urine,  and  then  in  very  small  quanti- 
ties. It  is  also  found  in  the  different  tissues  and  organs,  especially  abun- 
dant in  the  brain  and  the  nervous  system;  further,  in  the  yolk  of  the  egg, 
in  semen,  in  wool-fat  (together  with  isocholesterin),  and  in  sebum.  It 
appears  also  in  the  contents  of  the  intestine,  in  excrements,  and  in  the 
meconium.  It  especially  occurs  pathologically  in  gall-stones,  as  well  as  in 
atheromatous  cysts,  in  pus,  in  tuberculous  masses,  old  transudates,  cystic 
fluids,  sputum,  and  tumors.  It  does  not  exist  free  in  all  cases;  for  exam- 
ple, it  exists  in  part  as  fatty-acid  esters  in  wool-fat,  blood,  lymph,  brain 
vemix  caseosa,  and  epidermis  formations.  Several  kinds  of  cholesterin, 
called  phytosterines,  have  been  found  in  the  vegetable  kingdom. 

Cholesterin  which  has  been  crystallized  from  warm  alcohol  on  cooling 
and  that  which  is  present  in  old  transudates  contains  1  molecule  of  water  of 
crystallization,  melts  at  145°  C,  and  forms  colorless,  transparent  plates 
whose  sides  and  angles  frequently  appear  broken  and  whose  acute  angle  is 
often  76°  30'  or  87°  30'.  In  large  quantities  it  appears  as  a  mass  of  white 
plates  which  shine  like  mother-of-pearl  and  have  a  greasy  feeling. 

Cholesterin  is  insoluble  in  water,  dilute  acids,  and  alkalies.  It  is  neither 
dissolved  nor  changed  by  boiling  caustic  alkali.  It  is  easily  soluble  in  boil- 
ing alcohol  and  crystallizes  on  cooling.  It  dissolves  readily  in  ether, 
chloroform,  and  benzene,  and  also  in  the  volatile  or  fatty  oils.  It  is  dis- 
solved to  a  slight  extent  by  alkali  salts  of  the  bile-acids,  better  in  the  pres- 
ence of  oleic  soap  (Gerard  2),  The  solutions  in  ether  and  chloroform  are 
levorotatory. 

Among  the  many  compounds  of  cholesterin  studied  by  Obermuller 
the  propionic  ester  C2H5.CO.O.C27H45  is  of  special  interest  because  of  the 

'  Salkowski's  Festschrift,  1904,  and  Neuberg,  Ber.  d.  d.  chem.  Gesellsch.,  39. 
'  Compt.  rend.  soc.  biolog.,  58. 


336  THE  LIVER. 

behavior  of  the  fused  compound  on  cooUng,  and  it  is  used  in  the  detection 
of  cholesterin.  For  the  detection  of  cholesterin  use  is  made  of  its  reaction 
with  concentrated  sulphuric  acid,  which  gives  colored  products. 

If  a  mixture  of  five  parts  sulphuric  acid  and  one  part  water  acts  on 
a  cholesterin  crj'stal,  this  crystal  will  shov/  colored  rings,  first  a  bright 
carmine-red  and  then  violet.  This  fact  is  employed  in  the  microscopic 
detection  of  cholesterin.  Another  test,  and  one  verj^  good  for  the  micro- 
scopical detection  of  cholesterin,  consists  in  treating  the  crj-stals  first  with 
the  above  dilute  acid  and  then  with  some  iodine  solution.  The  crystals 
wi.l  be  gradually  colored  violet,  bluish  green,  and  a  beautiful  blue. 

Salkowski's  ^  Reaction.  The  cholesterin  is  dissolved  in  chloroform 
and  then  treated  with  an  equal  volume  of  concentrated  sulphuric  acid. 
The  cholesterin  solution  becomes  first  bluish  red,  then  gradually  more 
violet-red,  while  the  sulphuric  acid  appears  dark  red  with  a  greenish  fluor- 
escence. If  the  chloroform  solution  is  poured  into  a  porcelain  dish  it 
becomes  violet,  then  green,  and  finally  yellow. 

LiEBERiLA.Nx-BuRCH.A_RD's  ^  Reaction.  Dissolve  the  cholesterin  in  about 
2  c.c.  chloroform  and  add  first  10  drops  of  acetic  anhydride  and  then  con- 
centrated sulphuric  acid  drop  by  drop.  The  color  of  the  mixture  will 
first  be  a  beautiful  red,  then  blue,  and  finally,  if  not  too  much  cholesterin 
or  sulphuric  acid  is  present,  a  permanent  green.  In  the  presence  of  very 
little  cholesterin  the  green  color  may  appear  immediately, 

Neuberg  Rauchwerger's  ^  Reaction.  With  rhanmose,  or  better  still 
with  o-methylfurfurol  and  concentrated  sulphuric  acid,  an  alcoholic 
solution  of  cholesterin  gives  a  pink  ring,  or  after  mixing  the  liquids  and 
cooling,  a  pink  solution.  On  proper  dilution  an  absorption-band  can  be 
seen  just  beginning  before  E  and  whose  other  side  coincides  with  h.  This 
reaction  is  of  interest  because  it  is  also  given  by  bile-acids,  some  camphor 
derivatives,  abietinic  acid,  and  a  hydride  of  retene.  For  details  of  its 
performance,  see  original  publication. 

Pure,  dry  cholesterin  when  fused  in  a  test-tube  over  a  low  flame  with  two  or 
three  drops  of  propionic  anhydride  yields  a  mass  which  on  cooling  is  first  violet, 
then  blue,  green,  orange,  carmine-red,  and  finally  copper-red.  It  is  best  to  re-fuse 
the  mass  on  a  glass  rod  and  then  to  observe  the  rod  on  cooling,  holding  it  against 
a  dark  background  (Obermuller). 

Schiff's  Reaction.  If  a  little  cholesterin  is  placed  in  a  porcelain  dish  with 
the  addition  of  a  few  drops  of  a  mixture  of  2  or  .3  vols,  of  concentrated  hydrochloric 
acid  or  sulphuric  acid  and  1  vol.  of  a  rather  dilute  solution  of  ferric  chloride 
and  carefully  evaporated  to  dryness  over  a  small  flame,  a  reddish-violet  residue 

is  first  obtained  and  then  a  bluish- violet. 

*  Pfliiger's  Arch.,  6. 

'  C.  Liebermann,  Bar.  d.  deutsch.  chem.  Gesellsch.,  18,  1804;  H.  Burchard,  Bei- 
trage  zur  Kenntnis  der  Cholesterine.  Rostock,  1889. 

'  Salkowski's  Festschrift,  1904. 


CHOLESTEP.LX.  337 

If  a  small  quantity  of  cholesterin  is  evaporated  to  dryness  with  a  drop  of 
concentrated  nitric  acid,  one  obtains  a  yellow  spot  which  becomes  deep  orange-red 
with  ammonia  or  caustic  soda  (not  a  characteristic  reaction). 

Koprosterin  is  the  name  given  by  Bondzynski  to  the  cholesterin  which  was 
isolated  by  him  from  human  faeces,  although  it  was  prepared  earlier  by  Flint 
and  designated  as  stercorin.  It  dissolves  in  cold,  absolute  alcohol  and  very  readily 
in  ether,  chloroform,  and  benzene.  It  crystallizes  in  fine  needles  which  melt  at 
<)5-96°C.  (89-90°  according  to  Hausmanx),i  and  is  dextrorotatory,  (a)D= +24°. 
It  gives  the  same  color  reactions  as  cholesterin,  with  the  exception  that  it  does 
not  give  a  reaction  with  propionic  anhydride.  According  to  Bondzynski  and 
HuMNiCKi  it  is  a  dihydrocholesterin,  with  the  formula  C07H48O,  which  is  formed 
in  the  human  intestine  by  the  reduction  of  ordinary  cholesterin.  These  investi- 
gators have  found  another  cholesterin,  hippokoprosterin,  with  the  formula  C27H54O, 
in  horses'  faeces. 

Isocholesterin  is  a  cholesterin,  so  called  by  Schulze,^  with  the  formula 
C2«H430H,  which  occurs  in  wool-fat  and  is  therefore  found  to  a  great  extent  in 
so-called  lanolin.  It  gives  the  Liebermann-Burcharo  reaction,  but  does  not 
give  Salkowski's  reaction.     It  melts  at  138-138.5°  C. 

Spongosterin  is  the  name  givenby  Henze  ^  to  a  cholesterin  isolated   by  him 

from  a  silicious  sponge.     It  is  very  similar  to  cholesterin  but  is  not  identical  with 

it  or  with  phytocholesterins.     It  gives  the  Liebermann-Burchard  reaction  as 

well  as  Salkowski's  reaction,  but  the  last  test  is  not  quite  so  beautiful  a  red. 

25 
Obermijller's  reaction  is  negative.     Its  specific  rotation  is  («)^  =  19.59°. 

The  cholesterins  belong  to  the  so-called  lipoids,  which  have  been  men- 
tioned in  previous  chapters  (V  and  VI)  and  are  of  the  greatest  importance 
as  constituents  of  the  outer  envelope  of  erj'throcytes  and  the  cells  in 
general.  In  this  regard  the  cholesterin  is  of  special  interest  for  haemolysis, 
in  that,  as  shown  by  Ransom,  it  inhibits  the  hsemolytic  action  of  saponin 
and  hence  it  has  a  certain  protective  power  in  the  animal  body.  This 
action  of  cholesterin,  as  found  by  Haus\la.nn,  is  destroyed  by  replacing 
the  hydroxyl  groups.  According  to  Madsen  and  Noguchi  ^  the  combina- 
tion of  cholesterin  and  saponin  is  a  loose  one. 

The  so-called  cholesterin-stones  are  best  emplo3'ed  in  the  preparation 
of  cholesterin.  The  powder  is  first  boiled  with  water  and  then  repeatedly 
boiled  with  alcohol.  The  cholesterin  which  on  cooling  separates  from  the 
"warm  filtered  solution  is  boiled  with  a  solution  of  caustic  potash  in  alcohol 
so  as  to  saponify  any  fat.  After  the  evaporation  of  the  alcohol  the  choles- 
terin is  extracted  from  the  residue  with  ether,  by  which  the  soaps  are  not 

'  Bondzynski,  Ber.  d.  deutsch.  chera.  Gesellsch.,  29;  Bondzynski  and  Humnicki, 
Zeitschr.  f.  physiol.  Chem.,  22;  Flint,  ibid.,  23,  and  Amer.  Journ.  Med.  Sciences,  1862; 
Miiller,  Zeitsclir.  f.  physiol.  Chem.,  29;   Hausmann.  Hofmeister's  Beitrage,  6. 

^  Ber.  d.  deutsch.  chem.  Gesellsch.,  6;  Journal  f.  prakt.  Chem.  (N.  F.),  25;  and 
Zeitschr.  f.  physiol.  Chem.,  14,  522.  See  also  E.  Schulze  and  J.  Barbieri,  Journal  f. 
prakt.  Chem.  (N.  F.),2o,  159.  In  regard  to  the  formula  for  isocholesterin,  see  Darm- 
stadter  and  Lifschiitz,  Ber.  d.  deutsch.  chem.  Gesellsch.,  31,  and  E.  Schulze,  ibid.,  1200. 

^  Zeitschr.  f.  physiol.  Chem.,  41. 

♦Ransom,  Deutsch.  med.  Wochenschr.,  1901;  Hausmann,  Hofmeister's  Beitrage, 
6;  Madsen  and  Noguchi,  Kgl.  Dansk.  Vidensk.  Selskabs.  Forh.,  1904. 


338  THE  LIVER. 

dissolved;  filter,  evaporate  the  ether,  and  purify  the  cholesterin  by 
recrj'stallization  from  alcohol-ether.  The  cholesterin  may  be  extracted 
with  fat  from  tissues  and  fluids  by  first  extracting  with  ether  and  then 
proceeding  as  suggested  by  Ritter.i  The  essential  points  in  his  method 
consist  in  saponifying  the  fat  with  sodium  alcoholate,  removing  the  alcohol 
by  evaporating  to  dryness  with  NaCl,  and  finally  extracting  the  dried, 
pulverized  mass  with  ether.  After  evaporating  the  ether  the  residue  is 
dissolved  in  as  little  alcohol  as  possible  and  the  cholesterin  precipitated 
b}'  the  addition  of  water.  It  is  ordinarily  easily  detected  in  transudates 
and  pathological  formations  by  means  of  the  microscope. 

*  Zeitschr.  f.  physiol.  Chem.,  34. 


.CHAPTER  IX. 

DIGESTION. 

The  purpose  of  digestion  is  to  separate  those  constituents  of  the 
food  which  serve  as  the  nutriment  of  the  body  from  those  which  are  useless, 
and  to  separate  each  in  such  a  form  that  it  may  be  taken  up  by  the  blood 
from  the  alimentary  canal  and  employed  for  various  purposes  in  the 
organism.  This  demands  not  only  mechanical,  but  also  chemical  action. 
The  first  action,  wliich  is  essentially  dependent  upon  the  physical  properties 
of  the  food,  con  ists  in  a  tearing,  cutting,  crushing,  or  grinding  of  the  food,, 
while  the  second  serves  chiefly  in  converting  the  nutritive  bodies  into  a 
soluble  and  easily  absorbed  form,  or  in  splitting  them  into  simpler 
compounds  for  use  in  the  animal  syntheses.  The  solution  of  the  nutritive 
bodies  may  take  place  in  certain  cases  by  the  aid  of  water  alone,  but  in 
most  cases  a  chemical  metamorphosis  or  cleavage  is  necessary;  this  is 
effected  by  means  of  the  acid  or  alkaline  fluids  secreted  by  the  glands.  The 
study  of  the  processes  of  digestion  from  a  chemical  standpoint  must  there- 
fore begin  with  the  digestive  fluids,  their  qualitative  and  quantitative 
composition,  as  well  as  their  action  on  the  nutriments  and  foods. 

I.    The  Salivary  Glands  and  the  Saliva. 

The  salivary  glands  are  partly  albuminous  glands  (as  the  parotid  in  man 
and  mammals  and  the  submaxillary  in  rabbits),  partly  mucous  glands  (as 
some  of  the  small  glands  in  the  buccal  cavity  and  the  sublingual  and  sub- 
maxillary glands  of  many  animals),  and  partly  mixed  glands  (as  the  sub- 
maxillary gland  in  man).  The  alveoli  of  the  albuminous  glands  contain  cells 
which  are  rich  in  proteid  but  which  contain  no  mucin.  The  alveoli  of  the 
mucin-glands  contain  cells  rich  in  mucin  but  poor  in  proteid.  Cells  arranged 
in  different  ways,  but  rich  in  proteids,  also  occur  in  the  submaxillary  and 
sublingual  glands.  According  to  the  analyses  of  Oidtmann  ^  the  salivary 
glands  of  a  dog  contain  790  p.  m.  water,  200  p.  m.  organic  and  10  p.  m. 

»  Cit.  from  v.  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4.  Anfl.,  732.  The  figiires 
tliere  given  amount  to  1010  parts  instead  of  1000  parts. 

339 


340  DIGESTION. 

inorganic  solids.  Among  the  solids  we  find  mucin,  proteids,  nucleoproteids, 
nuclein,  enzymes  and  their  zymogens,  besides  extractive  bodies,  leucine,  xan- 
thine bodies,  and  mineral  substances. 

The  occurrence  of  a  mucinogen  has  not  been  proved.  On  the  complete  removal 
of  all  mucin  E.  Holmgren  '  found  no  mucinogen  in  the  submaxillary  gland  of  the 
ox,  but  a  mucin-like  gluconucleoproteid. 

The  saliva  is  a  mixture  of  the  secretion  of  the  above-mentioned  groups 
of  glands;  therefore  it  is  proper  that  a  study  be  made  of  each  of  the  differ- 
ent secretions  by  itself  and  then  of  the  mixed  saliva. 

The  submaxillary  saliva  in  man  may  be  easily  collected  by  introducing 
a  canula  through  the  papillary  opening  into  Wharton's  duct. 

The  submaxillary  saliva  has  not  always  the  same  composition  or  prop- 
erties; this  depends  essentially,  as  shown  by  experiments  on  animals,  upon 
the  conditions  under  which  the  secretion  takes  place.  That  is  to  say,  the 
secretion  is  partly  dependent  on  the  cerebral  system,  through  the  facial 
fibres  in  the  chorda  tympani,  and  partly  on  the  sympathetic  nervous  system, 
through  the  fibres  entering  the  vessels  in  the  gland.  In  consequence  of 
this  dependence  the  two  distinct  varieties  of  submaxillary  secretion  are 
distinguished  as  chorda-  and  sympathetic  saliva.  A  third  kind  of  saliva, 
the  so-called  paralytic  saliva,  is  secreted  after  poisoning  with  curare  or 
after  the  severing  of  the  glandular  nerves. 

The  difference  between  chorda-  and  sympathetic  saliva  (in  dogs)  con- 
sists chiefly  in  their  quantitative  constitution;  the  less  abundant  sym- 
pathetic saliva  is  more  viscous  and  richer  in  solids,  especially  in  mucin, 
than  the  more  abundant  chorda-saliva.  The  specific  gravity  of  the  chorda- 
saliva  of  the  dog  is  1.0039-1.0056,  and  contains  12-14  p.  m.  solids  (Eck- 
HARD^).  The  sympathetic  has  a  specific  gravity  of  1.0075-1.018,  with 
16-28  p.  m.  solids.  The  freezing-point  of  the  chorda-saliva  obtained  from 
dogs  on  electric  stimulation  varies,  according  to  Nolf,^  between  J=  —0.193° 
and  —0.396°,  with  a  content  of  3.3-6.5  p.  m.  salts  and  4.1-11.5  p.  m.  organic 
substances.  The  osmotic  pressure  is  on  an  average  a  little  higher  than 
one  half  the  osmotic  pressure  of  the  blood-serum.  The  spontaneously 
secreted  submaxillary  saliva  is  ordinarily  somewhat  diluted.  Other 
investigators,  such  as  Asher  and  Cutter,"*  have  also  found  that  the  osmotic 
pressure  of  the  submaxillary  saliva  is  considerably  lower  than  that  of  the 
blood.  The  gases  of  the  chorda-saliva  have  been  investigated  by  PFLtJGER.^ 
He  found  0.5-0.8  per  cent  oxygen,  0.9-1  per  cent  nitrogen,  and  64.73-85.13 

'  Upsala  Lakaref.  Forh.  (N.  F.),  2;   also  Maly's  Jahresber.,  27. 
'  Cited  from  Kijhne's  Lehrb.  d.  physiol.  Chem.,  7 
See  Maly's  Jahresber.,  31,  494. 

*  Zeitschr.  f.  Biologie,  40. 

*  Pfliiger's  Arch.,  1. 


PAROTID  SALIVA.  341 

per  cent  carbon  dioxide — all  results  calculated  at  0°  C.  and  760  mm.  pres- 
sure.    The  greater  part  of  the  carbon  dioxide  ^vas  chemically  combinetl. 

The  two  kinds  of  submaxillary  secretion  just  named  have  not  thus 
f^r  been  separately  studied  in  man.  The  secretion  may  be  excited  by  an 
emotion,  by  mastication,  and  by  irritating  the  mucous  membrane  of  the 
mouth,  especially  with  acid-tasting  substances.  The  submaxillary  saliva 
in  man  is  ordinarily  clear,  rather  thin,  a  little  ropy,  and  froths  easily.  Its 
reaction  is  alkaline  towards  litmus.  The  specific  gravity  is  1.002-1.003, 
and  it  contains  3.6-4.5  p.  m.  solids.^  As  organic  constituents  are  found 
mucin,  traces  of  proteid  and  diastatic  enzyme,  which  latter  is  absent  in 
several  species  of  animals.  The  inorganic  bodies  are  alkali  chlorides, 
sodium  and  magnesium  phosphates,  and  bicarbonates  of  the  alkalies  and 
calcium.     Potassium  sulphocyanide  occurs  in  this  saliva. 

The  Sublingual  Saliva.  The  secretion  of  this  saliva  is  also  influenced 
by  the  cerebral  and  the  sympathetic  nervous  system.  The  chorda-saliva, 
which  is  secreted  only  to  a  small  extent,  contains  numerous  salivary  cor- 
puscles, but  is  otherwise  transparent  and  very  ropy.  Its  reaction  is 
alkaline,  and  it  contains,  according  to  Heidexhain,-  27.5  p.  m.  solids  (in 
dogs). 

The  sublingual  secretion  in  man  is  clear,  mucilaginous,  more  alka- 
line than  the  submaxillary  saliva,  and  contains  mucin,  diastatic  enzyme^ 
and  potassium  sulphocyanide. 

Buccal  mucus  can  be  obtained  pure  from  animals  only  b}'  the  method 
suggested  by  Bidder  and  Schmidt,  which  consists  in  tying  the  exit  to  all 
the  large  salivar}-  glands  and  cutting  off  their  secretion  from  the  mouth. 
The  quantity  of  liquid  secreted  under  these  circumstances  (in  dogs)  was 
so  ver\^  small  that  the  investigators  named  were  able  to  collect  only  2 
grams  of  buccal  mucus  in  the  course  of  one  hour.  It  is  a  thick,  ropy,  sticky 
liquid  containing  mucin;  it  is  rich  in  form-elements,  above  all  in  flat  epi- 
thelium-cells, mucous  cells,  and  salivary  corpuscles.  The  quantity  of  solids 
in  the  buccal  mucus  of  the  dog  is,  according  to  Bidder  and  Schmidt,^ 
9.98  p.  m. 

Parotid  Saliva.  The  secretion  of  this  saliva  is  also  partly  dependent 
on  the  cereljral  nervous  system  (n.  glossopharyngeus)  and  parti}-  on  the 
sympathetic.  The  secretion  may  be  excited  by  emotions  and  by  irri- 
tation of  the  glandular  nerves,  either  directly  (in  animals)  or  reflexly.  by 
mechanical  or  chemical  irritation  of  the  mucous  membrane  of  the  mouth. 
Among  the  chemical  irritants  the  acids  take  first  place.     Mastication  also- 


'See  Maly,   "Chemie  der  Verdauungssafte  iind  der  Verdauung."  in  Hermann's 
Handb.,  5,  part  II,  18.     This  article  contains  also  the  pertinent  literature. 
2  Studien  d.  phyeiol.  Instituts  zu  Breslau,  Heft  4. 
^  Die  Verdauungssafte  und  der  Stoffwechsel  (Mitau  and  Leipzig,  1852),  5 


342  DIGESTION. 

exercises  a  strong  influence  upon  the  secretion  of  parotid  saliva,  which  is 
specially  marked  in  certain  herbivora. 

Human  parotid  saliva  may  be  readily  collected  by  the  introduction  of  a 
canula  into  Stexson's  duct.  This  saliva  is  thin,  less  alkaline  than  the 
submaxillary  saliva  (the  first  drops  are  sometimes  neutral  or  acid),  without 
special  odor  or  taste.  It  contains  a  little  proteid  but  no  mucin,  which  is  to 
be  expected  from  the  construction  of  the  gland.  It  also  contains  a  dias  atic 
enzyme,  which,  however,  is  absent  in  many  animals.  The  quantity  of  solids 
varies  between  5  and  16  p.  m.  The  specific  gravity  is  1.003-1.012.  Potas- 
sium sulphocyanide  seems  to  be  present,  though  it  is  not  a  con  .ant  con- 
stituent. KiJLz  1  found  a  maximum  of  1.46  p3r  cent  oxygen,  3.8  per  cent 
nitrogen,  and  in  all  66.7  per  cent  carbon  dioxide  in  human  parotid  saliva. 
The  quantity  of  firmly  combined  carbon  dioxide  was  62  per  cent. 

The  quantity  and  composition  of  the  saliva  from  the  mucin  glands  as 
well  as  from  the  albuminous  glands,  as  Pawlow's^  school  has  shown,  is 
greatl}'  dependent  in  dogs  upon  the  psychical  excitement,  but  also  upon  the 
kind  of  substances  introduced  into  the  mouth,  and  an  adaptation  of  the 
glands  for  various  mechanical  and  chemical  irritants  is  found  to  occur. 
Under  the  influence  of  hard  and  dry  food  the  glands  secrete  abundance  of 
saliva,  while  with  food  rich  in  water  the  secretion  is  considerably  less  and 
accommodates  itself  according  to  the  quantity  of  water  in  the  food.  Milk 
is  an  exception  to  this  rule,  as  it  causes  a  more  abundant  secretion  of  saliva 
than  meat.  This  is  of  importance  in  digestion  of  milk,  as  in  the  stomach 
the  mixture  of  milk  and  saliva  does  not  coagulate  to  a  compact  mass  but 
separates  in  a  finely  divided,  readily  digestible  condition.  By  the  action  of 
strong  chemical  bodies  the  saliva  is  secreted  in  proportion  to  the  strength 
of  the  irritant.  The  irritants  are  thereby  diluted  and  the  mouth  washed 
■out  at  the  same  time.  The  partaking  of  acids  brings  about  the  secre- 
tion of  a  thin  saliva,  poor  in  mucin,  in  quantities  sufficient  to  neutarl- 
ize  the  acid,  while  on  the  introduction  of  food  the  gland  i  secrete  a  saliva 
rich  in  mucin  and  diastatic  enzymes. 

The  mixed  buccal  saliva  in  man  is  a  colorless,  faintly  opalescent,  slightly 
ropy,  easily  frothing  liquid  w'ithout  special  odor  or  taste.  It  is  made  turbid 
by  epithelium-cells,  mucous  and  salivary  corpuscles,  and  often  by  food 
residues.  Like  the  submaxillary  and  parotid  saliva,  on  exposure  to  the  air 
It  becomes  covered  with  an  incrustation  consisting  of  calcium  carbonate  and 
a  small  quantity  of  an  organic  substance,  or  it  gradually  becomes  cloudy. 
Its  reaction  is  generally  alkaline  to  litmus.     The  degree  of  alkalinity  varies 

»  Zeitschr.  f.  Biologie,  23. 

^  Arch,  intemation.  de  Physiol.,  1, 1904.  See  also  Neilson  and  Terry,  Amer.  Journ. 
of  Physiol.,  15.  Somewhat  contradictory  statements  in  regard  to  the  accommoda- 
tion of  the  secretion  of  the  glands  to  requirements  (in  man)  can  be  found  in  Zebrovski, 
Pflijger's  Arch.,  110. 


PTYALIN.  343 

considerably  not  only  in  different  individuals  but  also  in  the  same  indi- 
vidual during  different  parts  of  the  day,  so  that  it  is  difficult  to  state  the 
average  alkalinity.  According  to  Chittenden  and  Ely  it  corresponds  to 
the  alkalinity  of  0.8  p.  m.  Na2C03  solution,  or  to  0.2  p.  m,  solution  accord- 
ing to  CoHN.  According  to  Foa  the  actual  alkalinity  ("OH-ion  concentra- 
tion) is  always  considerably  less  than  that  found  by  titration,  and  the 
reaction  determined  electrometrically  is  very  nearly  neutral.  The  reac- 
tion may  also  be  acid,  as  found  by  Sticker  to  be  the  case  some  time  after 
a  meal,  but  this  is  not  true  at  least  for  all  individuals.  The  specific  gravity 
varies  between  1.002  and  1.008,  and  the  quantity  of  solids  between  5 
and  10  p.  m.  According  to  Cohn  i,  J  =—0.20°  on  an  average  and  the 
amount  of  NaCl  is  1.6  p.  m.  The  solids,  irrespective  of  the  form-constitu- 
ents mentioned,  consist  of  'protein,  mucin,  oxidases,^  two  enzymes,  ptyalin 
and  maltase,  and  mineral  bodies.  It  is  also  claimed  that  urea  is  a  normal 
constituent  of  the  saliva.  The  min  ral  bodies  are  alkali  chlorides,  bicar- 
bonates  of  the  alkalies  and  calcium,  phosphates,  and  traces  of  sulphates, 
nitrites,  ammonia,  and  sulphocyanides,  which  latter  average  about  0.1 
p.  m.  (MuNK  and  others).  Smaller  quantities,  0.03-0.04  p.  m.,  are  found  in 
he  saliva  of  non-smokers  (Schneider  and  KRiJGER),  while  from  ordinary 
smokers  the  quantity  of  sulphocyanides  may  rise  to  0.2  p.  m.  (Fleck- 
seder  3), 

Sulphocyanides,  which,  although  not  constant,  occur  in  the  saliva  of 
man  and  certain  animals,  may  be  easily  detected  by  acidifying  the  saliva 
with  hydrochloric  acid  and  treating  with  a  very  dilute  solution  of  ferric 
chloride.  As  control,  especially  in  the  presence  of  very  small  quantities, 
it  is  best  to  compare  the  test  with  another  test-tube  containing  an  equal 
amount  of  acidulated  water  and  ferric  chloride.  Other  methods  have 
been  suggested  by  Gscheidlen,  Solera,  and  Ganassini.  The  quantita- 
tive estimation  can  be  done  according  to  Munk's  ^  method. 

Ptyalin,  or  salivarj^  diastase,  is  the  amylolytic  enzyme  of  the  saliva. 
This  enzyme  is  found  in  human  saliva,^  but  not  in  that  of  all  animals, 

'  Chittenden  and  Ely,  Amer.  Chem.  Joum.,  4,  1883;  Chittenden  and  Richards, 
Amer.  Joum.  of  Physiol.,  1;  Foa,  Compt.  rend.  ^c.  biolog.,  58;  Sticker,  cited  from 
Centralbl.  f.  Physiol.,  3,  237;  Cohn,  Deutsch.  med.  Wochenschr.,  1900. 

^  Bogdanow-Beresowski,  cited  from  Biochem.  Centralbl.,  2,  653. 

'Munk,  Virchow's  Arch.,  69;  Schneider,  Amer.  Journ.  of  Physiol.,  5;  Kriiger, 
Zeitschr.  f.  Biologie,  37;  Fleckseder,  Centralbl.  f.  innere  Med.,  1905.  lu  regard  to 
the  variation  in  the  amoimt  of  various  constituents  in  saliva  see  Fleckseder,  1.  c,  and 
Tezner,  Arch,  intemation.  de  Physiol.,  2. 

*  Gscheidlen,  Maly's  Jahresber.,  4;  Solera,  see  ibid.,  7  and  8;  Munk,  Virchow's 
Arch.,  69;   Ganassini,  Biochem.  Centralbl.,  2,  p.  361. 

'  In  regard  to  the  variation  in  the  quantity  of  ptyalin  in  himian  saliva  see   Hof- 
bauer,  Centralbl.  f.  Physiol.,  10,  and  Chittenden  and  Richards,  Amer.  Journ.  of  Physiol. 
1;   Schiile,  Maly's  Jahresber.,  29;  Tezner,  1.  c. 


344  DIGESTION. 

especially  not  in  the  typical  carnivora.  It  occurs  not  only  in  adults,  but 
also  in  new-bom  infants.  In  opposition  to  Zweifel's  views,  Berger  ^ 
claims  that  it  is  present  not  only  in  the  parotid  gland  of  children,  but  also 
in  the  mucin  gland. 

According  to  H.  Goldschmidt  ^  the  saliva  (parotid  saliva)  of  the  horse  does 
not  contain  ptyalin,  but  a  zymogen  of  the  same,  while  in  other  animals  and  man 
the  ptyalin  is  formed  from  the  zymogen  during  secretion.  In  horses  the  zymogen 
is  transformed  into  ptyalin  during  mastication,  and  bacteria  seem  to  give  the 
impulse  to  this  change.  During  precipitation  with  alcohol  the  zymogen  is  changed 
into  ptyalin. 

Ptyalin  has  not  been  isolated  in  a  pure  form  up  to  the  present  time.  It 
ran  be  obtained  purest  by  Cohnheim's^  method,  which  consists  in  carry- 
ing the  enzyme  down  mechanically  with  a  calcium-phosphate  precipitate 
and  washing  the  precipitate  with  water,  which  dissolves  the  ptyalin,  and 
from  which  it  can  l)e  obtained  by  precipitating  with  alcohol.  For  the 
study  or  demonstration  of  the  action  of  ptyalin  one  employs  a  watery  or 
glycerine  extract  of  the  salivary  glands,  or  simply  the  saliva  itself. 

Ptyalin,  like  other  enzymes,  is  characterized  by  its  action.  This  con- 
sists in  converting  starch  into  dextrins  and  sugar.  The  process  going  on 
in  this  conversion  may  be  described  as  follows:  In  the  first  stages  soluble 
starch  or  amidulin  is  formed.  From  this  amidulin,  er3-throdextrin  and 
sugar  are  produced  by  hydrolytic  cleavage.  The  erythrodextrin  then  splits 
into  a-achroodextrin  and  sugar.  From  this  achroodextrin  by  splitting 
^-achroodextrin  and  sugar  are  formed,  and  finally  this  /9-achroodextria 
splits  into  sugar  and  pachroodextrin.  Other  investigators  explain  this 
process  in  another  manner  (see  Chapter  III),  hence  the  exact  procedure  is 
not  completely  clear.  Still  the  results  are  positive  as  to  the  sugar  pro- 
duced in  this  process.  For  a  long  time  it  was  considered  that  dextrose  was 
the  sugar  formed  from  starch  and  glycogen,  but  Seegen  and  0.  Nasse  have 
shown  that  this  is  not  true.  Musculus  and  v.  Mering  have  shown  that 
the  sugar  formed  b}^  the  action  of  saliva,  amylopsin,  and  diastase  ujDon 
starch  and  glycogen  is  for  the  most  part  maltose.  This  has  been  substan- 
tiated by  Brown  and  Heron.  E.  Kulz  and  J.  Vogel  ^  have  also  demon- 
strated that  in  the  saccharification  of  starch  and  glycogen,  isomaltose, 
maltose,  and  some  dextrose  are  formed,  the  varying  quantities  depending 
upon  the  amount  of  ferment  and  the  length  of  its  action.     The  formation  of 


1  Zweifel,  Untersuchungen  iiber  den  Verdauungsapparat  der  Neugeborenen  (Berlin, 
1874);  Berger,  see  Maly's  Jahresber.,  30,  309. 

2  Zeitschr.  f .  physiol.  Chem.,  10. 

3  Virchow's  Arch.,  28. 

*  Seegen,  Centralbl.  f.  d.  med.  Wissensch.,  1876,  and  Pfliiger's  Arch.,  19;  Nasse 
ibid.,  ll;  Musculus  and  v.  Mering,  Zcitscl\r.  f.  physiol.  Chem.,  2;  Brown  and  Heron, 
Liebig's  Annal.,  199  and  204;   Kiilz  and  Vogel,  Zeitschr.  f.  Biologie,  31 


ACTION   OF   PTYALIN.  345 

dextrose  is  claimed  by  Tebb,  Rohmann,  and  Hamburger  ^  to  be  only  a 
product  of  the  inversion  of  the  maltose  by  the  maltase. 

The  action  of  ptyalin  in  various  reactions  has  been  the  subject  of  numer- 
ous invest igations.2  Natural  alkaline  saliva  is  very  active,  but  it  is  not 
so  active  as  when  made  neutral.  It  may  be  still  more  active  under  cer- 
tain circumstances  in  faintly  acid  reaction,  and  according  to  Chittenden 
and  Smith  it  acts  better  when  enough  hydrochloric  acid  is  added  to  satu- 
rate the  proteins  present  than  when  only  neutralized.  \Yhen  the  acid- 
combined  protein  exceeds  a  certain  amount,  then  the  diastatic  action  is 
diminished.  The  addition  of  alkali  to  the  saliva  decreases  its  diastatic 
action;  on  neutralizing  the  alkali  with  acid  or  carbon  dioxide  the  retarding 
or  preventive  action  of  the  alkali  is  arrested.  According  to  Schierbeck, 
carbon  dioxide  has  an  accelerating  action  in  neutral  liquids,  while  Ebstein 
claims  that  it  has,  as  a  rule,  a  retarding  action.  Organic  as  well  as  inorganic 
acids,  when  added  in  sufficient  quantity,  may  stop  the  diastatic  action 
entirely.  The  degree  of  acidity  necessary  in  this  case  is  not  always  the 
same  for  a  certain  acid,  but  is  dependent  upon  the  quantity  of  ferment. 
The  same  degree  of  acidity  in  the  presence  of  large  amounts  of  ferment  has 
a  weaker  action  than  in  the  presence  of  smaller  quantities.  Hydrochloric 
acid  is  of  special  physiological  interest  in  this  regard,  for  it  prevents 
the  formation  of  sugar  even  in  very  small  amounts  (0.03  p.  m.).  HyHro- 
chloric  acid  has  not  only  the  property  of  preA'enting  the  formation  of  sugar, 
but,  as  shown  by  Langley,  Nylen,  and  others,  may  entirely  destroy  the 
enzyme.  This  is  important  in  regard  to  the  physiological  significance  of 
the  saliva.  That  boiled  starch  (paste)  is  quickly,  and  unboiled  starch  only 
slowly,  converted  into  sugar  is  also  of  interest.  Various  kinds  of  unboiled 
starch  are  converted  with  different  degrees  of  rapidity. 

Several  series  of  investigations  have  been  made  upon  the  velocity  with 
which  ptyalin  acts,  and  as  in  testing  enzyme  action  in  general,  the  experi- 
menters have  not  made  use  of  the  different  times  required  to  produce  equal 
chemical  changes  as  a  measure  of  the  velocit}',  but  have  taken  the  quan- 
tities of  substance  changed  in  equal  times.  Although  the  results  are  some- 
what divergent  it  is  possible  to  deduce  the  following  from  them.  The 
velocity  increases,  at  least  under  conditions  othenvise  favorable,  with  the 
amount  of  enzyme  and  with  an  increasing  temperature  to  a  little  above  40°  C. 


^Tebb,  Journ.  of  Physiol.,  15;   Rohmann,  Ber.  d.  deutsch.   chem.  Gesellsch.,  27; 
Hamburger,  Pflager's  Arch. ,  60. 

2  See  Hammarsten,  Maly's  Jahresber.,  1;  Chittenden  and  Griswold,  Amer.  Chem. 
Jomn.,  3;  Langley,  Journal  of  Physiol.,  3;  Nylen,  Maly's  Jahresber.,  12,  241;  Chit- 
tenden and  Ely,  Amer.  Chem.  Journ.,  1;  Langley  and  Eves,  Journal  of  Physiol.,  4; 
Chittenden  and  Smith,  Y.ile  College  Studies,  1,  1885,  1;  Schlesinger,  Virchow's  Arch.. 
125;  Schierbeck,  Skand.  Arch.  f.  Physiol.,  3;  Ebstein  and  C.  Schulze,  Virchow's  Ari'h. 
134;   Kubcl.  Pfliiger's  Arch.,  76. 


346  DIGESTION. 

Foreign  substances,  such  as  metallic  salts,i  have  different  effects.  Certain 
salts  even  in  small  quantities  completely  arrest  the  action;  for  example, 
HgCl2  accomplishes  this  result  completely  by  the  presence  of  only  0.05  p.  m 
Other  salts,  such  as  magnesium  sulphate,  in  small  quantities  (0.25  p.  m.) 
accelerate,  and  in  larger  quantities  (5  p.  m.)  check  the  action.  The  presence 
of  peptone  has  an  accelerating  action  on  the  sugar  formation  (Chittenden 
and  Smith  and  others).  The  accumulation  of  the  products  of  the  amyloiytic 
decomposition  also  checks  the  action  of  the  saliva.  This  has  been  shown  by 
special  experiments  made  by  Sh.  Lea.^  He  made  parallel  experiments 
with  digestions  in  test-tubes  and  in  dialyzers,  and  found  on  the  removal  of 
the  products  of  the  amyloiytic  decomposition  by  dialysis  that  the  forma- 
tion of  sugar  took  place  sooner,  but  also  that  considerably  more  maltose 
and  less  dextrin  were  formed. 

To  show  the  action  of  saliva  or  ptyalin  on  starch  the  three  ordinary 
tests  for  dextrose  may  be  used,  namely,  Moore's  or  Trommer's  test  or 
the  bismuth  test  (see  Chapter  XV).  It  is  also  necessar}^,  as  a  control,  to 
first  t€st  the  starch-paste  and  the  saliva  for  the  presence  of  dextrose.  The 
steps  in  he  transformation  of  starch  into  amidulin,  erythrodextrin,  and 
achroodextrin  may  be  shown  by  testing  with  iodine. 

Maltose  occurs  in  saliva  to  only  a  slight  extent.  It  converts  maltose 
into  dextrose.  According  to  Sticker  3  saliva  also  has  the  power  of  splitting 
sulphuretted  hydrogen  from  the  sulphur  oils  of  radishes,  onions,  and  cer- 
tain other  vegetables. 

The  quantitative  composition  of  the  mixed  saliva  must  vary  considerably, 
not  only  because  of  individual  differences,  but  also  because  under  varying 
conditions  there  may  be  an  unequal  division  of  the  secretion  from  the 
different  glands.  We  give  opposite  a  few  analyses  of  human  saliva  as 
examples  of  its  composition.     The  results  are  in  parts  per  1000. 

Hammerbacher  found  in  1000  parts  of  the  ash  from  human  saliva:  potash 
457.2,  soda  95.9,  iron  oxide  50.11,  magnesia  1.55,  sulphuric  anhydride  (SO3)  63.8, 
phosphoric  anhydride  (P2O3)  188.48,  and  chlorine  183.52. 

The  quantity  of  saliva  secreted  during  twenty-four  hours  cannot  be  ex- 
actly determined,  but  has  been  calculated  by  Bidder  and  Schmidt  to  be 
1400-1500  grams.  The  most  abundant  secretion  occurs  during  meal-times. 
According  to  the  calculations  and  determinations  of  Tuczek  *,  in  man  1 
gram  of  gland  yields  13  grams  of  secretion  in  the  course  of  one  hour  during 
mastication.     These  figures  correspond  fairly  well  with  those  representing 

*  See  O.  Xasse,  Pfliiger's  Arch.,  11,  and  Chittenden  and  Painter,  Yale  College 
Studies,  1,  1885,  52;   Kubel,  Pfliiger's  Arch.,  76. 

'  Joum.  of  Physiol.,  11. 

*  Miinch.  med.  >/ochenschr.,  43. 

♦Bidder  and  Schmidt,  1.  c,  13;  Tuczek,  Zeitschr.  f.  Biologie,  12. 


COMPOSITION   OF  THE   SALIVA. 


347 


the  average  secretion  from  1  gram  of  gland  in  animals,  namely,  14.2  grams 
in  the  horse  and  8  grams  in  oxen.  The  quantity  of  secretion  per  hour 
may  be  8  to  14  times  greater  than  the  entire  mass  of  glands,  and  there  is 
probably  no  gland  in  the  entire  body,  so  far  as  is  known  at  present — the 
kidneys  not  excepted — whose  ability  of  secretion  under  physiological  con- 


* 

p 

s 

m 

X 

o 
a 
p 
o 

■< 

1-5 

IS 

a 
o 
S 

fa 

•V 

c 

si 

<  a 

u 

H 

a 

a 

X 

z 
z 
■< 
s 

a 

►J 

Water 

992.9 

7.1 

1.4 

3.8 

995 . 16 

4.84 

1.62 
1.34 

0.06 
1.82 

994.1 
5.9 

2.13 
1.42 

0.10 
2.19 

988.3 
11.7 

994.7 
5.3 

3;5-8'4 

in 
filtered 
.saliva. 

994  2 

Solids 

5  8 

Mucus  and  epithelium 

2.2 

Soluble  organic  substances . 

(Ptyalin  of  early  investigators.) 

Sulphocyanides 

3.27 

0.064 

to 
0.090 

1.4 
0.04 

Salts 

1.9 

1.30 

2.2 

ditions  equals  that  of  the  salivary  glands.  A  remarkably  abundant  secre- 
tion of  saliva  is  induced  by  pilocarpine,  while  atropine,  on  the  contrary, 
prevents  it. 

That  the  secretion  of  saliva,  even  if  we  do  not  consider  such  substances 
as  ptyalin,  mucin,  and  the  like,  is  not  a  process  of  filtration,  follows  from 
many  reasons,  especially  the  following:  The  salivary  glands  have, 
a  specific  property  of  eliminating  certain  substances,  such  as  potas- 
sium salts  (Salkowski  ^),  iodine,  and  bromine  compounds,  but  not  others, 
for  example,  iron  compounds  and  dextrose.  It  is  also  noticeable  that 
the  saliva  is  richer  in  solids  when  it  is  eliminated  quickly  by  gradually  in- 
creased stimulation,  and  in  larger  quantities  than  when  the  secretion  is 
slower  and  less  abundant  (Heidenhain).  The  amount  of  salts  increases 
also  to  a  certain  degree  by  an  increasing  rapidity  of  elimination  (Heiden- 
hain, Werther,  Laxgley  and  Fletcher,  Novi  ^). 

Like  the  secretion  processes  in  general,  the  secretion  of  saliva  is  closely 
connected  with  the  processes  in  the  cells.  The  chemical  processes  going  on 
in  these  cells  during  secretion  are  still  unknown. 

'  Zeitschr.  f.  physiol.  Chem.,  5.  The  other  analyses  are  cited  from  Maly,  Chemie 
der  Verdauungssafte,  Hermann's  Handbuch  d.  Physiol.,  5,  part  II,  14. 

^  Virchow's  Arch.,  53. 

3  Heidenhain,  Pfliiger's  Arch.,  17;.  Werther,  ibid.,  38;  Langley  and  Fletcher, 
Proc.  Roy.  Soc,  45,  and  especially  Phil.  Trans.  Roy.  Soc.  London,  180;  Novi, 
Arch.  f.  (Anat.  u.)  Phsyiol.,  1888. 


348  DIGESTION. 

The  Physiological  Importance  of  the  Saliva.  The  quantity  of  water  in 
the  saUva  renders  possible  the  action  of  certain  bodies  on  the  organs  of 
taste,  and  it  also  serves  as  a  solvent  for  a  part  of  the  nutritive  substances. 
The  importance  of  the  saliva  in  mastication  is  especially  marked  in  her- 
bivora,  and  there  is  no  question  as  to  its  importance  in  facilitating  the  act 
of  swallowing.  The  saliva  containing  mucin  is  especially  important  in 
this  regard,  and  Pawlow's  school  has  shown  that  the  secretion  also  regu- 
lates itself  in  this  regard.  The  saliva  is  also  of  importance,  as  it  serves  in 
washing  out  the  mouth  and  thereby  acts  as  a  protection  against  destructive 
substances  or  bodies  foreign  to  the  mouth.  The  power  of  converting  starch 
into  sugar  is  not  inherent  in  the  saliva  of  all  animals,  and  even  when  it  pos- 
sesses this  property  the  intensity  varies  in  different  animals.  In  man, 
whose  saliva  forms  sugar  rapidly,  a  production  of  sugar  from  (boiled)  starch 
undoubtedly  takes  place  in  the  mouth,  but  how  far  this  action  proceeds 
after  the  morsel  has  entered  the  stomach  depends  upon  the  rapidit}^  with 
which  the  acid  gastric  juice  mixes  with  the  swallowed  food,  and  also  upon 
the  relative  amounts  of  the  gastric  juice  and  food  in  the  stomach.  The 
large  quantity  of  water  which  is  swallowed  with  the  saliva  must  be  ab- 
sorbed and  pass  into  the  blood,  and  it  must  in  this  way  go  through  an 
intermediate  circulation  in  the  organism.  Thus  the  organism  possesses  in 
the  saliva  an  active  medium  by  which  a  constant  stream,  convejdng  the 
dissolved  and  finely  divided  bodies,  passes  into  the  blood  from  the  intestinal 
canal  during  digestion. 

Salivary  Concrements.  The  so-called  tartar  is  yellow,  gray,  yellowish-gray, 
brown  or  black,  and  has  a  stratified  structure.  It  may  contain  more  than  200  p.  m. 
organic  substances,  which  consist  of  mucin,  epithelium,  and  leptothrix-chains. 
The  chief  part  of  the  inorganic  constituents  consists  of  calcium  carbonate  and 
phosphate.  The  salivary  calculi  may  vary  in  size  from  that  of  a  small  grain  to 
that  of  a  pea  or  still  larger  (a  salivary  calculus  has  been  found  weighing  18.6 
grams),  and  they  contain  variable  quantities  of  organic  substances  (50-380  p.m.), 
which  remain  on  extracting  the  calculus  with  hydrochloric  acid.  The  chief  in- 
organic constituent  is  calcium  carbonate. 


II.    The  Glands  of  the  Mucous  Membrane  of  the  Stomach,  and  the 

Gastric  Juice. 

Since  long  ago  the  glands  of  the  mucous  coat  of  the  stomach  have  been 
divided  into  two  distinct  classes.  Those  which  occur  in  the  greatest  abun- 
dance and  which  have  the  greatest  size  in  the  fundus  are  called  fundus  glands, 
also  rennin  or  pepsin  glands,  and  the  others  which  occur  only  in  the  neigh- 
borhood of  the  pylorus  have  received  the  name  of  pyloric  glands,  sometimes 
also,  though  incorrectly,  called  mucous  glands.  The  division  of  these  two 
forms  of  glands  in  the  mucous  membrane  of  the  stomach  is  essentially 
different  in  various  animals.     The  mucous  coating  of  the  stomach  is  cov- 


GASTRIC    JUICE.  349 

ered  throughout  with  a  layer  of  columnar  epithelium,  which  is  generally 
considered  as  consisting  of  goblet  cells  that  produce  mucus  by  a  metamor- 
phosis of  the  protoplasm. 

The  fundus  glands  contain  two  kinds  of  cells:  adelo.morphic  or  chief 
cells,  and  delomorphic  or  parietal  cells,  the  latter  formerly  called 
REXxix  or  pepsin  cells.  Both  kinds  consist  of  protoplasm  rich  in  proteins; 
but  their  relationship  to  coloring-matters  seems  to  show  that  the  protein 
substances  of  both  are  not  identical.  The  nucleus  must  consist  chiefly  of 
nuclein.  Besides  the  above-mentioned  constituents,  the  fimdus  glands 
contain  as  more  specific  constituents  several  enz3'mes  or  their  zymogens, 
besides  a  little  fat  and  cholesterin. 

The  pyloric  glands  contain  cells  which  are  generally  considered  as 
related  to  the  above-mentioned  chief  cells  of  the  fundus  glands.  As  these 
glands  were  formerly  thought  to  contain  a  larger  quantity  of  mucin,  they 
were  also  called  mucous  glands.  According  to  Heidexhaix,  independent 
of  the  columnar  epithelium  of  the  excretory  ducts  they  take  no  part  worthy 
of  mention  in  the  formation  of  mucus,  which  according  to  his  views  is 
effected  by  the  epithelium  covering  the  mucous  membrane.  The  pyloric 
glands  also  "seem  to  contain  the  zymogens  referred  to  above.  Alkali  chlo- 
rides, alkali  phosphates,  and  calcium  phosphates  are  found  in  the  mucous 
coating  of  the  stomach. 

LiEBERMANX  ^  has  obtained  an  acid-reacting  residue  on  digesting  the  mucosa 
of  the  stomach  with  pepsin-h^'drochlorie  acid,  which  strangely  enough  contained  no 
nuclein,  but  only  a  protein  containing  lecithin,  called  lecithalbumin.  To  this 
lecithalbumin  he  ascribes  a  great  importance  in  the  secretion  of  hydrochloric  acid. 

The  Gastric  Juice.  The  observations  of  Helm  and  Beaumoxt  on  per- 
sons with  gastric  fistula  led  to  the  suggestion  that  gastric  fistulas  be  made 
on  animals,  and  this  operation  was  first  performed  by  Bassow  -  in  1842  on 
a  dog.  Verxeuil  performed  the  same  on  a  man  in  1876  with  successful 
results.  Pawlow^^  has  recently  improved  the  surger}'  of  gastric  fistula 
and  has  added  much  to  the  study  of  gastric  secretion. 

The  secretion  of  gastric  juice  is  not  continuous,  at  least  in  man  and  in 
the  mammals  experimented  upon.  It  only  occurs  under  psychic  influence, 
and  also  by  stimulation  of  the  mucous  membrane  of  the  stomach  or  the 
intestine.  The  most  exhaustive  researches  on  the  secretion  of  gastric  juice 
(in  dogs)  have  been  made  by  PA^^'Low  and  his  pupils. 

'  Pfliiger's  Arch.,  50. 

'Helm,  Zwei  Krankengeschichten,  Wien,  1803.  cit.  from  Hermann's  Handbuch, 
5.  part  II,  39;  Beaumont,  '"The  Physiologj'  of  Digestion,"  1833;  Bassow,  Bull,  de 
la  SCO.  des  natur.  de  Moscou,  16,  cit.  from  Maly  in  Hermarm's  Handbuch,  5,  38; 
Temeuil,  see  Ch.  Richet,  "Du  .sue  gastrique  chez  Thomme,"  etc.  (Paris,  1878),  158. 

'  Pawlow,  Die  Arbeit  der  Verdauungsdriisen  (Wiesbaden,  1898),  where  the  works 
of  his  pupils  are  also  mentioned.     See  also  Ergebnisse  der  Physiologic,  1,  Abt.  1. 


350  DIGESTION. 

In  order  to  obtain  gastric  juice  free  from  saliva  and  food  residues  they  arranged 
besides  a  gastric  fistula  also  an  oesophageal  fistula  from  which  the  swallowed  food 
could  be  withdrawn  with  the  saliva  without  entering  the  stomach,  and  in  this 
manner  an  apparent  feeding  was  possible.  In  this  way  it  was  possible  to  study 
the  influence  of  psychical  moments  on  one  side  and  the  direct  action  of  food  on 
the  mucous  membrane  on  the  other.  After  a  method  suggested  by  Heidenhain 
and  later  improved  by  Pawlow  and  Chigin,  they  have  succeeded  in  preparing  a 
blind  sac  by  partial  dissection  of  the  fundus  part  of  the  stomach,  and  the  secretion 
processes  could  be  studied  in  this  sac  while  the  digestion  in  the  other  parts  of  the 
stomach  was  going  on.  In  this  way  they  were  able  to  study  the  action  of  different 
foods  on  the  secretion. 

The  most  essential  results  of  the  investigations  of  Pawlow  and  his 
pupils  are  as  follows:  Mechanical  stimulation  of  the  mucosa  does  not  pro- 
duce any  secretion.  Chemical  and  mechanical  irritations  of  the  mucous 
membrane  of  the  mouth  cause  no  reflex  excitation  of  the  secretory  nerves 
of  the  stomach.  There  are  two  moments  which  cause  a  secretion,  namely, 
the  psychical  moment — the  passionate  desire  for  food  and  the  sensation  of 
satisfaction  and  pleasure  on  partaking  it — and  the  chemical  moment,  the 
action  of  certain  chemical  substances  on  the  mucous  membrane  of  the 
stomach.  The  first  moment  is  the  most  important.  The  secretion  occur- 
ring under  its  influence  by  the  vagus  fibres  appears  earlier  than  that  pro- 
duced by  chemical  irritants,  but  only  after  an  interval  of  at  least  4|  minutes. 
This  secretion  is  more  abundant  but  less  continuous  than  the  "chemical." 
It  yields  a  more  acid  and  active  juice  than  the  latter.  As  chemical  excitants 
which  cause  a  secretion  reflexively  through  the  stomach  mucosa  we  include 
only  water  and  certain  unknown  extractive  substances  contained  in  meat 
and  meat  extracts,  in  impure  peptone,  and  also,  it  seems,  in  milk.  Accord- 
ing to  Herzex  and  Radzikowski  ^  alcohol  is  also  a  strong  agent  in  pro- 
ducing a  flow  of  juice.  Carbonated  alkalies  have  a  preventive  instead  of 
an  accelerating  action  on  secretion.  Bitter  substances  partaken  of  in 
small  amounts  a  certain  time  before  a  meal  increase  the  secretion,  while 
larger  amounts  have  a  retarding  action  (Bomssow,  Strashesko  2).  Fats 
have  a  retarding  action  on  the  appearance  of  secretion  and  diminish  the 
quantity  of  juice  secreted  as  well  as  the  amount  of  enzyme.  The  sub- 
stances, such  as  egg-albumin,  which  act  as  chemical  stimulants  cannot  be 
digested  by  the  "psychical"  secretion,  but  may  perhaps  cause  a  chemical 
secretion  b}^  their  decomposition  products. 

The  quantity  of  juice  secreted  during  digestion  is  proportional  to  the 
quantity  of  food,  and  the  secretion  of  gastric  juice  may  also  be  influenced 
by  the  kind  of  food.  This  action  of  various  foods — meat,  bread,  and  milk — 
may  be  arranged  in  progressive  series  as  follows: 

*  Pfliiger's  Arch.,  84,  513. 

'  Borissow,  Arch.  f.  exp.  Path.  u.  Pharm.,  51;  Strashesko,  see  Biochem.  Centralbl., 
4c  148. 


SECRETION  OF   GASTRIC   JUICE.  351 


Acidity. 

Digestive  Activity. 

Duration  of  Secretion. 

1. 

Meat. 

Bread. 

Bread. 

2. 

Milk. 

Meat. 

Meat. 

3. 

Bread. 

Milk. 

Milk. 

The  acidity  is  greatest  with  a  meat  diet  and  lowest  with  bread;  the 
quantity  of  enzyme  is,  on  the  contrary,  highest  with  a  bread  diet  and 
lowest  with  milk. 

The  secretion  in  the  stomach  may  also  be  influenced  by  the  small  in- 
testine, and  in  this  way,  as  shown  by  the  investigations  of  Pa^^xow  and 
his  pupils,  the  fats  have  a  retarding  action  upon  the  secretion  of  juice  and 
upon  digestion  by  acting  reflexly  upon  the  duodenal  mucosa.  In  dogs  on 
feeding  fat  (oil)  with  food  containing  starch,  the  secretion  of  gastric  juice 
remains  reduced  during  the  entire  period  of  feeding,  and  fat  in  connection 
with  protein  food  has  a  similar  action,  with  the  exception  that  in  this  case 
the  retarding  action  is  observed  only  in  the  first  hours  of  digestion.  Ac- 
cording to  PioxTKOWSKi  1  the  oil-soaps  differ  from  the  neutral  fats  by 
having  a  strong  action  on  the  flow  of  juice,  and  this  is  the  reason  why  about 
5  to  6  hours  after  a  meal  with  fat  the  secretion  of  juice  is  stopped,  as  just 
at  this  time  the  soaps  are  being  formed.  According  to  Frouix  the  food 
in  the  intestine  produces  a  secretion  of  gastric  juice  which  continues  after 
the  action  of  the  psychic  moment  has  ceased.  Leconte^  arrived  at  sim- 
ilar results,  and  he  ascribes  less  importance  to  the  chemical  secretion  as  com- 
pared with  the  psychic  secretion  than  Pawlow  does. 

LoNNQUiST^  has  made  observations  upon  dogs  as  to  the  dependence 
of  the  secretion  of  gastric  juice  upon  the  presence  of  food  or  substances 
causing  flow  in  the  stomach  or  in  the  intestine  alone,  or  simultaneously  in 
both,  w'ith  the  exclusion  of  the  psychical  influence.  For  this  purpose  he 
expsrimented  with  the  stomach,  isolated  according  to  Heidexhaix-Paw- 
Low,  as  well  as  with  fistulse  in  the  stomach  and  intestine,  and  besides  this 
he  also  separated  the  stomach  and  intestine  from  each  other  by  means 
of  a  membrane  between  the  pylorus  and  the  intestine  produced  by  oper- 
ative means.  An  abundant  secretion  of  juice  was  produced  in  the  stomach 
isolated  from  the  intestine  by  water,  alcohol,  meat,  and  meat  extracts, 
and  by  the  digestive  products  of  egg-albumin.  Dilute  hydrochloric  acid 
(0.1-0.5  per  cent)  or  natural  gastric  juice  caused  only  a  faint  secretion. 
Dilute  sodium-chloride  or  soda  solutions  (0.25-€.50  per  cent)  acted  some- 
what like  water;  stronger  soda  solutions  (1-1.5  per  cent)  produced  a  much 
greater  secretion  of  juice.  Water  or  salt  solution  in  the  duodenum  was 
without  action.     Liquid  fat  had  (reflexly)  a  strong  retarding  action,  and 

>  See  Biochem.  Centralbl.,  3,  660. 

'  Frouin,  Compt.  rend.  soc.  biol.,  53;   Leconte,  La  Cellule,  1". 

^  "Bidrag  tils  Kaimedomen  om  mags'aftafsondringen,"  Akademisk  afhandling.  Hel- 
siHgfors,  1906. 


352  DIGESTION. 

soda  solutions,  in  the  same  manner,  had  a  weaker  retarding  action.  Water 
as  well  as  alcohol  was  absorbed  from  the  stomach,  and  the  alcohol  acted 
for  the  first  half  hour  as  a  strong  excitant  for  the  flow  of  juice. 

We  know  very  little  positively  in  regard  to  the  gastric  secretion  in  man. 
According  to  the  older  statements  the  irritants  may  be  mechanical,  thermic, 
and  chemical.  Among  the  chemical  excitants  we  include  alcohol  and  ether, 
which  in  too  great  a  concentration  bring  about  no  physiological  secretion, 
rather  the  transudation  of  a  neutral  or  faintly  alkaline  fluid.  Certain  acids, 
such  as  carbonic  acid,  neutral  salts,  meat  extracts,  spices,  and  other  bodies 
also  belong  to  this  group.  The  statements  on  this  subject  are  unfortu- 
nately very  uncertain  and  contradictory,  still  there  is  no  doubt  that  in 
man,  at  least,  alcohol  and  meat  extracts  may  be  active  in  causing  secre- 
tion. 

The  question  as  to  how  far  the  observations  made  by  Pawlow  and  his 
school  can  be  applied  to  man  is  of  special  interest;  still  we  have  only  very 
few  statements  on  this  point  at  the  present  time.  Hornborg,^  who  recently 
studied  a  case  of  gastric  fistula  with  oesophageal  stricture  in  a  boy,  could 
not  observe  any  special  influence  of  the  psychic  excitement.  The  chewing 
of  indifferent  or  bad  tasting  bodies  had  no  action,  while  on  the  contrary 
the  chewing  of  bodies  with  a  pleasant  taste  produced  a  more  or  less  abun- 
dant secretion.  Umber  ^  observed  in  only  one  instance,  in  a  case  of  a 
man  after  gastrotomy,  an  insignificant  truly  psychic  secretion  of  gastric 
juice;  chewing  of  an  indifferent  body  or  of  chewing-tobacco  brought 
about  no  secretion.  After  an  apparent  feeding  with  meat,  with  a  latent 
period  of  3  minutes,  a  secretion  of  gastric  juice  rich  in  HCl  and 
enzymes  occurred.  Contrary  to  the  behavior  in  dogs,  the  juice  secreted 
after  chewing  bread  was  richer  in  acid  than  after  chewing  meat;  the 
quantity  was  on  the  contrary  less.  Umber  also  observed  that  after  the 
introduction  of  a  food  enema  into  the  rectum  a  secretion  of  gastric  juice 
was  produced  by  reflex  action.  Cade  and  Latarjet^  have  made  observa- 
tions on  a  girl  twenty  years  old  w^ho  had  a  blind  sac  formed  by  con- 
striction, which  was  analogous  to  Pawlow's  "little  stomach."  The  juice 
which  flowed  from  the  fistula  opening  of  this  blind  sac  was  at  least  not 
always  normal,  judging  from  the  amount  of  acid  and  by  its  action.  In 
this  person  a  purely  psychic  secretion  was  unquestionably  observed  by  a  con- 
tinuous recollection  of  a  pleasant  sensation  of  taste,  although  it  was  not 
especially  strong. 

From  these  observations  of  Hornborg  and  Umber,  as  well  as  from  some 

>  Hornborg,  "Bidrag  till  kannedomen  om  magsaftafsondringen  hos  manniskan  " 
Inaug.-Dissert.  Helsingfors,  1903. 
'Berlin,  klin.  Wochenschr.,  1905. 
'Compt.  rend.  soc.  biolog.,  57. 


COMPOSITION   OF  GASTRIC    JUICE,  3J3 

older  observations  of  Schule,  Troller,  Riegel,  and  Scheuer/  we  con- 
clude that  in  man  the  psychic  secretion  is  much  less  than  that  produced  b}- 
the  introduction  of  food  or  bodies  having  a  pleasant  taste.  That  the  prepar- 
ation of  the  food  in  the  mouth  has  an  essential  influence  upon  the  secretion 
is  proven  without  doubt,  but  we  are  not  united  as  to  how  this  action  takes 
place.  Certain  experimenters  consider  the  secreted  and  swallowed  saliva 
as  the  most  essential  factor  in  this  action,  while  others  believe  that  the  act 
of  chewing,  and  still  others  that  the  chemical  action  and  the  sense  of  taste, 
are  the  most  important. 

The  Qualitative  and  Quantitative  Composition  of  the  Gastric  Juice.  The 
human  gastric  juice,  which  can  seldom  be  obtained  pure  and  free  from 
residues  of  the  food  or  from  mucus  and  saliva,  is  a  clear,  or  only  ver\'  faintly 
cloudy,  and  in  man  nearly  colorless  fluid  of  an  insipid,  acid  taste  and  strong 
acid  reaction.  It  contains,  as  form-elements,  glandular  cells  or  their  nuclei, 
mucus-coripuscles,  and  more  or  less  changed  columnar  epithelium. 

The  acid  reaction  of  the  gastric  juice  depends  on  the  presence  of  free 
acid,  which,  as  has  been  learned  from  the  investigations  of  C.  Schmidt, 
RiCHET,  and  others,  consists,  when  the  gastric  juice  is  pure  and  free  from 
particles  of  food,  chief!}'  or  in  large  part  of  hydrochloric  acid.  Coxtejean  2 
has  regularly  found  traces  of  lactic  acid  in  the  pure  gastric  juice  of  fasting 
dogs.  After  partaking  of  food,  especially  after  a  meal  rich  in  carboh^'drates, 
lacnc  acid  occurs  abundantly,  and  sometimes  acetic  and  butyric  acids.  In 
new-bom  dogs  the  acid  of  the  stomach  is  lactic  acid,  according  to  Gmelin.3 
The  quantity  of  free  hydrochloric  acid  in  the  gastric  juice  is,  according  to 
Pawlow  and  his  pupils,  in  dogs  5-6  p.  m..  and  in  cats  an  average  of  5.20 
p.  m.  HCl.  In  man  (he  acidity  has  been  found  to  vary  considerably,  but 
it  is  generally  calculated  as  2-3  p.  m.  HCl.  Accorchng  to  Verhaegex's 
researches  there  is  no  doubt  that  pure  human  gastric  juice  from  perfectly 
healthy  persons  has  a  higher  acidity.  Umber  observed  after  apparent 
feeding  with  bread  3.5  p.  m.,  and  Horxborg*  found  higher  results  in  a 
boy  with  gastric  fistula.  The  juice  secreted  before  taking  food  contained 
3.05  p.  m.  acid.  After  taking  food  the  acidity  was  higher.  The  acidity 
of  juice  after  bread  was  3.65-5.11  p.  m.,  with  an  average  of  4.39  p.  m.,  and 
the  juice  after  meat  4.01-5.66  p.  m.,  or  an  average  of  4.62  p.  m.  There  is 
hardly  any  doubt  that  at  least  a  part  of  the  hydrochloric  acid  of  the  gastric 
juice  does  not  exist  free  in  the  ordinary  sense,  but  combined  with  organic 

'  The  literature  may  be  found  in  Umber's  work,  1.  c. 

^  Bidder  and  Schmidt,  Die  Verdauungssafte,  etc.,  44;  Richet,  1.  c;  Contejean,  Con- 
tributions a  I'etude  de  la  physiol.  de  I'estomac,  Theses,  Paris,  1892. 

'  Pfliiger's  Arch.,  90  and  103. 

*  See  Richet,  1.  c;  Contejean,  1.  c:  Verhaegen,  "La  Cellule,"  1896  and  1897;  Um- 
ber, 1.  c;  Hornborg,  1.  c;  and  the  literature  on  the  estimation  of  hydrochloric  acid 
in  the  gastric  contents  (p.  375). 


354  DIGESTIOX. 

substances.  The  results  obtained  for  the  acidity  of  gastric  juice  by 
physical  methods  are  nearly  identical  with  those  obtained  by  titration 
(P.  Fraxckel  1). 

As  chief  organic  constituent,  perfectly  fresh  gastric  juice  (of  dogs)  con- 
tains a  very  complex  substance  (or  perhaps  a  mixture  of  substances)  which 
coagulates  on  boiling  and  which  separates  on  strongly  cooling  the  juice. 
This  substance  is  considered  by  certain  experimenters  (Nexcki  and  Sieber, 
and  Pawlow)  as  the  conveyor  of  the  several  ferment  actions  of  the  gastric 
juice,  i.e.,  the  pepsin  as  well  as  the  rennin  action.  Gastric  juice  also  con- 
tains lecithin  and  chlorine,  and  yields  nucleoproteid.  proteose,  nuclein  bases, 
and  pentose  as  cleavage  products  (Nencki  and  Sieber-). 

The  specific  gravit}-  of  gastric  juice  is  low,  1.001-1.010.  It  is  corre- 
spondingly poor  in  solids.  Older  analyses  of  gastric  juice  from  man,  the  dog, 
and  the  sheep  were  made  by  C.  Schmidt.^  As  these  analyses  refer  only 
to  impure  gastric  juice  they  are  of  little  value.  The  quantity  of  solids 
in  saliva-free  gastric  juice  from  a  dog  was  27  p.  m.,  with  17.1  p.  m.  or- 
ganic substance.  The  quantity  of  free  hydrochloric  acid  was  3.1  p.  m. 
Besides  these  Schmidt  found  NaCl  1.46;  CaCU  0.6;  KCl  1.1;  NH4CI  0.5; 
earthy  phosphates  1.9;  and  FeP04  0.1  p.  m.  Nencki  ^  found  5  milli- 
grams sulphocyanic  acid  per  litre  of  gastric  juice  of  a  dog.  The  pure 
gastric  juice  of  another  dog  contained,  according  to  Nexcki  and  Sieber,- 
an  average  of  3.06  p.  m.  solids. 

Besides  the  free  hydrochloric  acid,  pepsin,  rennin,  and  a  lipase  are  the 
other  physiologically  important  constituents  of  gastric  juice. 

Pepsin.  This  enzyme  is  found,  with  the  exception  of  certain  fishes,  in 
all  vertebrates  thus  far  investigated. 

Pepsin  occurs  in  adults  and  in  new-born  infants.  This  condition  is 
different  in  new-born  animals.  Wliile  in  a  few  herbivora,  such  as  the 
rabbit,  pepsin  occurs  in  the  mucous  coat  before  birth,  this  enzyme  is 
entirely  absent  at  the  birth  of  those  carnivora  which  have  thus  far  been 
examined,  such  as  the  dog  and  cat. 

In  various  invertebrates  enzj^mes  have  also  been  found  which  have  a 
proteolytic  action  in  acid  solutions.  It  has  been  shown  that  these  enzymes, 
nevertheless,  are  not  in  all  animals  identical  with  ordinary  pepsin.  Accord- 
ing to  Klug  and  Wroblewski  ^  the  pepsins  found  in  man  and  various 
higher  animals  are  somewhat  different.  Enzymes  also  occur  in  various 
plants  and  animal  organs;  although  not  identical  with  pepsin,  they  act  i:i 
acid  reaction  and  stand  to  a  certain  extent  between  pepsin  and  trypsin. 

*  Zeitschr.  f.  exp.  Path.  u.  Therap.,  1. 
2  Zeitschr.  f.  physiol.  Chem.,  32. 

M.  c. 

*Ber.  d.  d.  chem.  Gesellsch.,  28. 

*  Klug,  Pfliiger's  Arch.,  60;  Wroblewski,  Zeitschr.  f.  physiol.  Chem.,  21. 


PEPSIN.  355 

To  this  group  belongs  Glaessner's  pseudopepsin,  which  according  to  him 
is  the  only  peptic  enzyme  in  the  pyloric  end.  Pseudopepsin,  whose  exist- 
ence is  disputed  by  Klug,  while  others  (Reach,  Pekelharixg)  affirm  its 
occurrence  in  the  mucous  membrane,  acts,  according  to  Glaessner,  also 
in  neutral  and  alkaline  reaction  and  yields  trs-ptophane  among  other  cleavage 
products.  According  to  Berg.maxx  ^  it  is  identical  with  erepsin  (see 
below).  Among  the  enzymes  of  the  mucosa  of  the  stomach  belongs  the 
so-called  antipepsin  discovered  by  Weixlaxd,^  which  has  a  retarding 
action  upon  pepsin  digestion  and,  as  some  claim,  prevents  the  self -digest  ion 
of  the  mucous  membrane. 

Pepsin  is  as  difficult  to  isolate  in  a  pure  condition  as  other  enzymes. 
The  pepsin  prepared  by  Brucke  and  Suxdberg  gate  negative  results  with 
most  reagents  for  proteins,  and  showed  nevertheless  a  powerful  action, 
which  seems  to  indicate  that  it  was  very  pure.  Schoumow-Simaxowski, 
Nexcki  and  Sieber,  and  also  Pekelharixg  have  designated  as  the  true 
enzyme  the  substance  containing  chlorine,  which  they  obtained  by  strongly 
cooling  the  gastric  juice.  That  this  substance  is  not  an  individual,  and 
hence  cannot  be  pepsin,  follows  from  the  investigations  of  Pekelharixg. 
WTiile  pepsin,  according  to  Nencki  and  Sieber.  was  rich  in  phosphorus 
and  contained  nucleoproteid,  Pekelharixg's  pepsin  was  free  from  phos- 
phorus and  yielded  no  nucleoproteid.  Friedexthal  and  Miva.mota^  have 
also  shown  that  the  pepsin  is  still  active  after  the  splitting  off  of  the  nuclein 
complex  (and  also  the  protein).  As  pepsin  is  readily  precipitated  with  the 
proteins  and  combines  therewith,  it  is  difficult  to  decide  whether  pepsin  is 
a  protein  substance  or  not,  and  the  question  as  to  the  nature  of  pepsin  is 
still  undecided,  just  as  is  the  case  with  other  enzymes.  As  ordinarily 
known,  pepsin,  at  least  in  an  impure  form,  is  soluble  in  water  and  glycerine. 
It  is  precipitated  by  alcohol,  but  only  slowly  destroyed  thereby.  In 
aqueous  solution  its  action  is  quickly  destroyed  on  heating  to  boiling. 
According  to  Bierxacki-^  pepsin  in  neutral  solutions  is  destroyed  by  heat- 
ing to  55°  C.  In  the  presence  of  2  p.  m.  HCl  a  temperature  of  55°  C.  is 
not  injurious,  and  the  compound  with  acid  is  more  resistant  than  the 
free  pepsin  (Grober  ^).  Pepsin  in  acid  solution  is  destroyed  b}-  heating 
to  65°  C.  for  five  minutes.     On  adding  peptone  and  certain  salts  the  pepsin 

^  Glaessner,  Hofmeister's  Beitrage,  1;  Ivlug,  Pfliiger's  Arch.,  92;  Reach,  Hofmeis- 
ter's  Beitrage,  4;  Pekelharing,  Arch,  des  scienc.  biolog.,  St.  Petersbourg,  11;  Pawlow- 
Festband,  1904;  Bergmann,  Skand.  Arch.  f.  Physiol.,  18. 

-  Zeitschr.  f.  Biologie,  44. 

'  Brucke,  Wien.  Sitzungsber.,  43;  Sundberg,  Zeitschr.  f.  physiol.  Chem.,  9;  Schou- 
mow-Simanowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  33;  Pekelharing,  Zeitschr.  f.  physiol. 
Chem..  22  and  35;  Xencki  and  Sieber,  ibid.,  32;  Friedenthal  and  Miyamota,  CentralbL 
f.  Physiol..  15,  785. 

*  Zeitschr.  f.  Biologie,  28. 

^  Arch.  f.  exp.  Path.  u.  Pharm.,  51. 


356  DIGESTION. 

may  be  heated  to  70°  C.  without  decomposing.  In  the  dry  state  it  can,  on 
the  contrary,  be  heated  to  over  100°  C.  without  losing  its  physiological 
action.  The  only  property  which  is  characteristic  of  pepsin  is  that  it  dis- 
solves protein  bodies  in  acid  but  not  in  neutral  or  alkaline  solutions,  with 
the  formation  of  proteoses,  peptones,  and  other  products. 

The  methods  for  the  preparation  of  relatively  pure  pepsin  depend,  as  a 
rule,  upon  its  property  of  being  thrown  down  with  finely  divided  precipi- 
tates of  other  bodies,  such  as  calcium  phosphate  or  cholesterin.  The 
rather  complicated  methods  of  BrIjcke  and  Sundberg  are  based  upon 
this  property.  Pekelharing  makes  use  of  a  prolonged  dialysis  and  pre- 
cipitation with  0.2  p.  m.  HCl. 

Very  permanent  pepsin  solutions,  from  which  the  enzyme  with  con- 
siderable protein  can  be  precipitated  by  alcohol,  may  be  prepared  by 
extraction  with  glycerine.  Solutions  having  a  strong  action  may  also  be 
prepared  l)y  making  an  infusion  of  the  gastric  mucosa  of  an  animal  in  acid- 
ified water  (2-5  p.  m.  HCl).  This  is  unnecessary,  as  we  can  obtain  pure 
gastric  juice  according  to  Pawlow's  method,  and  also  because  very  active 
commercial  preparations  of  pepsin  can  be  bought  in  the  market. 

The  Action  of  Pepsin  on  Proteins.  Pepsin  is  inactive  in  neutral  or 
alkaline  reactions,  but  in  acid  liquids  it  dissolves  coagulated  protein 
bodies.  The  protein  always  swells  and  becomes  transparent  before  it  dis- 
solves. Unboiled  fibrin  swells  up  in  a  solution  containing  I  p.  m.  HCl, 
forming  a  gelatinous  mass,  and  does  not  dissolve  at  ordinary  temperature 
within  a  couple  of  days.  Upon  the  addition  of  a  little  pepsin,  however, 
this  swollen  mass  dissolves  quickly  at  ordinary  temperatures.  Hard- 
boiled-egg  albumin,  cut  in  thin  pieces  with  sharp  edges,  is  not  perceptibly 
changed  by  dilute  acid  (2-4  p.  m.  HCl)  at  the  temperature  of  the  body  in 
the  course  of  several  hours.  But  the  simultaneous  presence  of  pepsin 
causes  the  edges  to  become  clear  and  transparent,  blunt  and  swollen,  and 
the  protein  gradually  dissolves. 

From  what  has  been  said  above  in  regard  to  pepsin,  it  follows  that 
proteins  may  be  employed  as  a  means  of  detecting  pepsin  in  liquids.  Ox- 
fibrin  may  be  employed  as  well  as  coagulated  egg-albumin,  which  latter  is 
used  in  the  form  of  slices  with  sharp  edges.  As  the  fibrin  is  easily  digested 
at  the  normal  temperature,  while  the  pepsin  test  with  egg-albumin  requires 
the  temperature  of  the  body,  and  as  the  test  with  fibrin  is  somewhat  more 
delicate,  it  is  often  preferred  to  that  with  egg-albumin.  When  we  speak  of 
the  "pepsin  test"  without  further  explanation,  we  ordinarily  understand 
it  as  the  test  with  fibrin. 

This  test,  nevertheless,  requires  care.  The  fibrin  used  should  be  ox- 
fibrin  and  not  pig-fibrin,  which  last  is  dissolved  too  readily  with  dilute  acid 
alone.  The  unboiled  ox-fibrin  may  be  dissolved  by  acid  alone  without 
pepsin,  but  this  generally  requires  more  time.  In  testing  with  unboiled 
fibrin  at  normal  temperature,  it  is  advisable  to  make  a  control  test  with 
another  portion  of  the  same  fibrin  with  acid  alone.  Since  at  the  tempera- 
ture of  the  body  unboiled  fibrin  is  more  easily  dissolved  by  acid  alone,  it  is 
best  always  to  work  with  boiled  fibrin. 


ESTIMATION   OF  PEPSIX.  357 

As  pepsin  has  not  thus  far  been  prepared  in  a  positively  pure  condition, 
it  is  impossible  to  determine  the  absolute  quantity  of  pepsin  in  a  liquid.  It 
is  only  possible  to  compare  the  relative  amounts  of  pepsin  in  two  or  more 
liquids,  which  may  be  done  in  several  ways. 

The  older  method,  that  of  Brucke,  consists  in  diluting  the  two  pepsin  solu- 
tions to  be  compared  with  certain  proportions  of  1  p.  m.  hydrochloric  acid,  so 
that  when  the  amount  of  pepsin  contained  in  the  original  solution  is  equal  to  1, 
each  solution  contains  a  degree  of  dilution,  p,  corresponding  to  1,  h,  \,  \,  ^,  etc. 
A  flock  of  fibrin  or  a  piece  of  hard-boiled  egg  is  added  to  each  test  and  the  time 
noted  when  each  test  begins  to  digest  and  when  it  ends.  The  relative  amount 
of  pepsin  is  calculated  from  the  rapidity  of  digestion  as  follows:  the  tests  p  =  i, 
i,  fs,  of  one  series  is  digested  in  the  same  time  as  tests  p=l,  ^,1  oi  the  other 
series;  hence  the  first  solution  contained  four  times  as  much  pepsin.  This  method 
is  not  used  as  often  as  the  following: 

Mett's  Method.  Draw  up  white  of  egg  m  a  glass  tube  1-2  millimetres  in  diam- 
eter, coagulate  it  by  plunging  it  into  hot  water  at  95°  C,  and  cut  the  ends  off 
sharply;  then  add  two  tubes  to  each  test-tube  with  a  few  cubic  centimetres  of  the 
acid  pepsin  solution;  allow  them  to  digest  at  body  temperature,  and  after  a  certain 
time,  generally  after  ten  hours,  measure  the  lineal  extent  of  the  digested  layer 
of  albumin  in  the  various  tests,  bearing  in  mind  that  the  digested  layer  at  each 
end  must  not  be  longer  than  6-7  millimetres.  The  quantity  of  pepsin  in  the 
comparative  tests  is  as  the  square  of  the  millimetres  of  the  albumin-column  dis- 
solved in  the  same  time.  Thus  if  in  one  case  2  millimetres  of  albumin  were  dis- 
solved and  in  the  other  3  millimetres,  then  the  quantity  of  pepsin  is  as  4:9.  If 
the  fluid  removed  from  the  stomach,  which  is  rich  in  bodies  ha"ving  a  disturbing 
influence  upon  pepsin  digestion,  is  to  be  tested,  then  the  liquid  must  be  first  prop- 
erly diluted  with  2-4  p.  m.  hydrocliloric  acid  (Xierexsteix  and  Schiff')- 

Objections  have  been  .'aised  against  these  methods  from  several  sides,  es- 
pecially by  Grxjtzxer,  but  they  can  be  recommended  for  practical  purposes  as 
being  simple  and  rather  accurate.  Huppert  and  E.  Schl'tz  measure  the  relative 
quantities  of  pepsin  from  the  amount  of  secondary  proteoses  formed  under  certain 
conditions.  The  proteoses  were  determined  by  the  polariscope.  J.  Schutz 
determines  the  total  proteose-nitrogen,  and  Spriggs  ■  finds  that  the  change  in  the 
viscosity  is  a  measure  of  the  amount  of  pepsin. 

VoLHARD  and  LoHLEiN  ^  use  an  acid  casein  solution  for  the  pepsin  determina- 
tion, and  determine,  after  precipitation  with  sodium  sulphate,  the  acidity  of  the 
filtrate  of  the  digested  test  as  weU  as  of  the  original  control  solution.  The  casein  is 
precipitated  as  an  acid  compound  by  the  sulphate,  and  the  filtrate  separated  from 
the  precipitate  contains  less  acid  than  the  original  solution.  In  proportion  as  the 
digestion  progresses  less  substance  is  precipitated  by  the  sulphate,  and  the  acidity 
of  the  fUtrate  becomes  correspondingly  higher.  The  increase  in  acidity  in  the 
different  portions  varies  withia  certain  limits  as  the  square  root  of  the  quantity 
of  ferment. 

GRtJTZNER's*  test  is  based  on  the  use  of  finely  cut  fibrin  colored  with  carmine. 
The  fibrin  is  first  allowed  to  sweU  up  in  1  p.  m.  hydrochloric  acid  and  then  about 
equal  quantities  are  placed  in  test-tubes  of  the  same  diameter  and  treated  with 
15  c.c.  of  1  p.  m.  hydrochloric  acid,     -\fter  the  addition  of  the  pepsin  solution  to 

'  Mett,  see  Pawlow,  1.  c,  31;   Xierenstein  and  Schiff,  Berl.  klin.  Wochenschr..  40. 

^  Huppert  and  Schiitz,  Pfliiger's  Arch.,  SO;  J.  Schiitz,  Zeitschr.  f.  physiol.  Chem., 
30;  Spriggs,  ibid.,  35. 

^  Hofmeister's  Beitrage,  7. 

*  Griitzner,  Pfliiger's  Arch.,  8  and  106.  See  also  A.  Kom,  •  Uber  Methoden  Pepsin 
quantitativ  zu  bestlmmen,"  Inaug.-Dis.sert.,  Tubingen    1902. 


358  DIGESTION. 

be  tested  the  fibrin  dissolves,  giving  a  red  color  to  the  solution,  and  the  strength 
of  the  digestion  is  determined  by  the  depth  of  the  color.  For  comparison  a  series 
of  tubes  are  used  containing  carmine  solution  diluted  in  known  proportions,  and 
which  are  arranged  so  that,  for  example,  when  one  pepsin  solution  had  a  color 
corresponding  to  No.  1,  and  the  other,  on  the  contrary,  to  No.  4,  then  the  latter 
had  four  times  as  much  fibrin  dissolved  as  the  first. 

The  rabidity  of  the  pepsin  digestion  depends  on  several  circumstances. 
Thus  different  acids  are  unequal  in  their  action;  hydrochloric  acid  shows 
in  slight  concentration,  0.8-1.8  p.  m.,  a  more  powerful  action  than  any  other 
acid,  whether  inorganic  or  organic.  In  greater  concentration  other  acids 
may  have  a  powerful  action;  but  no  constant  relationship  has  been  found 
between  the  strength  of  various  acids  and  their  action  in  pepsin  digestion, 
and  the  statements  in  regard  to  the  action  of  different  acids  are  somewhat 
contradictory.!  Sulphuric  acid,  it  seems,  has  a  weaker  action  than  the 
other  inorganic  acids.  The  degree  of  acidity  is  also  of  the  greatest  impor- 
tance. With  hydrochloric  acid  the  degree  of  acidity  is  not  the  same  for 
different  protein  bodies.  For  fibrin  it  is  0.8-1  p.  m.,  for  myosin,  casein,  and 
vegetable  proteins  about  1  p.  m.,  for  coagulated  egg-albumin,  on  the  con- 
trary, about  2.5  p.  m.  The  rapidity  of  the  digestion  increases,  at  least  to  a 
certain  point,  with  the  quantity  of  pepsin  present,  unless  the  pepsin  added 
is  contaminated  by  a  large  quantity  of  the  products  of  digestion,  which 
may  prevent  its  action. 

According  to  E.  ScHiJTZ,^  whose  statements  have  been  confirmed  ])y 
several  others,  the  digestion  products  produced  in  a  certain  time  are,  within 
certain  limits,  proportional  to  the  square  root  of  the  relative  amounts 
of  pepsin  (the  ScHiJTz-BoRissow^  rule).  The  accumulation  of  products 
of  digestion  has  a  retarding  action  on  digestion,  although,  according  to 
Chittenden  and  Amerman,^  the  removal  of  the  digestion  products  by  means 
of  dialysis  does  not  essentially  change  the  relationship  between  the  proteo.ses 
and  true  peptones.  Pepsin  acts  more  slowly  at  low  temperatures  than 
it  does  at  higher  ones.  It  is  even  active  in  the  neighborhood  of  0°  C,  but 
digestion  takes  place  very  slowly  at  this  temperature.  With  increasing 
temperature  the  rapidity  of  digestion  also  increases  until  about  40°  C, 
when  the  maximum  is  reached.  According  to  the  investigations  of  Flaum  * 
it  is  probable  that  the  relationship  between  proteoses  and  peptones  remains 
the  same,  irrespective  of  whether  the  digestion  takes  place  at  a  low^  or  high 
temperature,  as  long  as  the  digestion  is  continued  for  a  long  enough  time. 

*  See  Wroblewski,  Zeitschr.  f.  physiol.  Chem.,  21,  and  especially  Pfleiderer,  Pfliiger's 
Arch.,  66,  which  also  gives  references  to  other  works;  Larin,  Biochem.  Centralbl., 
1,  484;   and  A.  Pick,  Wien.  Sitzungsber.,  M.  N.  Klasse,  112. 

'  Zeitschr.  f.  physiol.  Chem.,  9. 
'  Journ.  of  PhysioL,  14. 

*  Zeitschr.  f.  Biologie,  28. 


ACTION  OF  PEPSIN.  359 

If  the  swelling  up  of  the  protein  is  prevented,  as  by  the  addition  of  neutral 
salts,  such  as  NaCl,  in  sufficient  amounts,  or  by  the  addition  of  bile  to  the 
acid  liquid,  digestion  can  be  prevented  to  a  greater  or  less  extent.  Foreign 
bodies  of  different  kinds  produce  different  actions,  in  which  naturally  the 
variable  quantities  in  which  they  are  added  are  of  the  greatest  importance- 
Salicylic  acid  and  carbolic  acid,  and  especially  sulphates  (Pfleiderer), 
retard  digestion,  while  arsenious  acid  promotes  it  (Chittenden),  and  hydro- 
cyanic acid  is  relatively  indifferent.  By  experiments  with  salt  solutions  so 
strongly  diluted  that  the  action,  on  account  of  the  strong  dissociation,  was 
brought  about  by  ions  and  not  by  the  electrolytically  neutral  molecules 
(min.  j\  and  max.  J  normal  salt  solutions),  J.  SchIjtz  i  found  that  the 
ani.ons  had  a  much  greater  retarding  action  upon  pepsin  digestion  than 
the  cations.  Of  these  latter  the  sodium  cation  had  the  strongest  retarding 
action.  Alcohol  in  large  quantities  (10  per  cent  and  above)  disturbs  the 
digestion,  while  small  quantities  act  indifferently.  Metallic  salts  in  very- 
small  quantities  may  indeed  sometimes  accelerate  digestion,  but  otherwise 
they  tend  to  retard  it.  The  action  of  metallic  salts  in  different  cases  can 
he  explained  in  various  ways,  but  they  often  seem  to  form  with  proteins 
insoluble  or  difficultly  soluble  combinations.  The  alkaloids  may  also 
retard  the  pepsin  digestion  (Chittenden  and  Allen  2).  A  very  large 
number  of  observations  have  been  made  in  regard  to  the  action  of  foreign 
substances  on  artificial  pepsin  digestion,  but  as  these  observations  have 
not  given  any  direct  result  in  regard  to  the  action  of  these  same  substances 
on  natural  digestion,  as  well  as  upon  secretion  and  absorption,  we  will  not 
discuss  them  here. 

The  Products  of  the  Digestion  of  Proteins  by  Means  of  Pepsin  and  Acid. 
In  the  digestion  of  nucleoproteids  or  nucleoalbumins  an  insoluble  residue 
of  nuclein  or  pseudonuclein  always  remains,  although  under  certain  cir- 
cumstances a  complete  solution  may  occur.  Fibrin  also  yields  an  insoluble 
residue,  which  consists,  at  least  in  great  part,  of  nuclein,  derived  from  the 
form-elements  enclosed  in  the  blood-clot.  This  residue  which  remains 
after  the  digestion  of  certain  proteins  was  called  dyspeptone  by  Meissner. 
This  name  is  therefore  not  only  unnecessary  but  indeed  erroneous,  as  this 
residue  does  not  consist  of  bodies  related  to  the  peptones.  In  the  digestion 
of  proteins,  substances  similar  to  acid  albuminates,  parapeptone  (Meiss- 
ner 3),  antialburnate,  and  antialbumid  (KitHxNe),  may  also  be  formed. 
On  separating  these  bodies  the  filtered  liquid,  neutralized  at  boiling-point, 


'  Hofmeister's  Beitriige,  5. 

^  Studies  from  the  Lab.  Physiol.  Chem.  Yale  University,  1,  76.  See  also  Chitten- 
den and  Stewart,  ibid.,  3,  60. 

^  The  works  of  Meissner  on  pepsin  digestion  are  found  in  Zeitschr.  f.  rat.  Med.,  7, 
8,  10,  12,  and  14. 


360  DIGESTION. 

contains  proteoses  and  peptones  in  the  old  sense,  while  the  sa-called  Kuhne 
true  peptone  and  the  other  cleavage  products  are  obtained  only  after  a 
longer  and  more  intense  digestion.  The  relationship  between  the  various 
proteoses  changes  very  much  in  different  cases  and  in  the  digestion  of  the 
various  proteins.  For  instance,  a  larger  quantity  of  primary  proteoses  is 
obtained  from  fibrin  than  from  hard-boiled-egg  albumin  or  from  the  pro- 
teins of  meat;  and  the  different  proteins,  according  to  the  researches  of 
Klug,^  yield  on  pepsin  digestion  unequal  quantities  of  the  various  digestive 
products.  In  the  digestion  of  unboiled  fibrin  an  intermediate  product 
may  be  obtained  in  the  earlier  stages  of  the  digestion — a  globulin  which 
coagulates  at  55°  C.  (Hasebroek  ^).  For  information  in  regard  to  the 
different  proteoses  and  peptones  which  are  formed  in  pepsin  digestion  the 
reader  is  referred  to  previous  pages  (50  to  61). 

Action  of  Pepsin-Hydrochloric  Acid  on  Other  Bodies.  The  gelatine- 
forming  substance  of  the  connective  tissue,  of  the  cartilage,  and  of  the  bones, 
from  which  last  the  acid  dissolves  only  the  inorganic  substances,  is  con- 
verted into  gelatine  by  digesting  with  gastric  juice.  The  gelatine  is  further 
changed  so  that  it  loses  its  property  of  gelatinizing  and  is  converted  into 
gelatoses  and  peptone  (see  page  79).  True  mucin  (from  the  submaxil- 
lary) is  dissolved  by  the  gastric  juice,  yielding  substances  similar  to  pep- 
tone and  a  reducing  substance  similar  to  that  obtained  by  boiling  with 
a  mineral  acid.  Mucoids  from  tendons,  cartilage,  and  bones  dissolve, 
according  to  Posxer  and  Gies,^  in  pepsin-hydrochloric  acid,  but  leave  a 
residue  which  amounts  to  about  10  per  cent  of  the  original  material  and 
which,  as  it  seems,  consists  in  great  part,  if  not  entirely,  of  a  combination  of 
proteid  with  glucothionic  acid  (Chapters  VII  and  VIII).  The  solution 
contains  primary  and  secondary  mucoproteoses  and  mucopeptones.  The 
former  contain  glucothionic  acid,  but  the  latter  do  not.  Elastin  is  dissolved 
more  slowly  and  yields  the  above-mentioned  substances  (page  76).  Ker- 
atin and  the  epidermal  formations  are  insoluble.  The  nucleins  are  dissolved 
with  difficulty,  and  the  cell  nuclei,  therefore,  remain  in  great  part  undis- 
solved in  the  gastric  juice.  The  animal  cell-membrane  is,  as  a  rule,  more 
easily  dissolved  the  nearer  it  stands  to  elastin,  and  it  dissolves  with  greater 
difficulty  the  more  closely  it  is  related  to  keratin.  The  membrane  of  the 
plant-cell  is  not  dissolved.  Oxyhcemoglobin  is  changed  into  hsematin  and 
protein,  the  latter  undergoing  further  digestion.  It  is  for  this  reason  that 
blood  is  changed  into  a  dark-brown  mass  in  the  stomach.  The  gastric 
juice  does  not  act  upon  fat,  but,  on  the  contrary,  dissolves  the  cell-membrane 
of  fatty  tissue,  setting  the  fat  free.     Gastric  juice  has  no  action  on  starch 


'  Pfliiger's  Arch.,  65. 

'  Zeitschr.  f.  physiol.  Chem.,  11. 

^  Amer.  Journ.  of  Physiol.,  11. 


CHY^IOSIX   AXD  PARACHYMOSIN.  361 

or  the  simple  varieties  of  sugar.  The  statements  in  regard  to  the  ability 
of  gastric  juice  to  invert  cane-sugar  are  vers'  contradictor}-.  At  least  this 
action  of  the  gastric  juice  is  not  constant,  and  if  it  is  present  at  all  it  is 
probably  due  to  the  action  of  the  acid. 

Pepsin  alone,  as  above  stated,  has  no  action  on  proteins,  and  an  acid  of  the 
intensity  of  the  gastric  juice  can  only  very  slowly,  if  at  all,  dissolve  coagulated 
albumin  at  the  temperature  of  the  body.  Pepsin  and  acid  together  not  only  act 
more  quickly,  but  qualitatively  they  act  otherwise  than  the  acid  alone,  at  least 
upon  dissolved  protein.  This  has  led  to  the  assumption  of  the  presence  of  a 
pepsin-hydrochloric  acid  whose  existence  and  action  are  only  hypothetical.  As 
pepsin  digestion,  it  seems,  j'ields  finally  the  same  products  as  the  hydrolytic 
cleavage  with  acids,  we  can  say  for  the  present  only  that  this  enz\Tne  acts  like 
other  catalyzers  in  very  powerfully  accelerating  a  process  which  would  proceed 
also  without  the  catalyzers. 

Chymosin  (rexxin)  and  Parachymosin.  So  far  two  different  rennet 
enzymes  have  been  obtained  from  animal  stomachs,  namely,  the  enzyme 
called  rennin  (chymosin),  which  is  found  in  the  calf's  stomach  and  has 
been  known  for  a  long  time,  and  the  parach3'mosin  discovered  by  Baxg^ 
which  is  the  rennet  enzyme  of  the  human  stomach.  This  latter  occurs  in 
the  gastric  juice  of  man  under  physiological  conditions,  but  may  be  absent 
under  special  pathological  conditions  (Schumburg,  Boas.  Johxsox',  Klem- 
perer2).  Chymosin  is  habitually  found  in  the  neutral,  waten,-  infusion  of 
the  fourth  stomach  of  the  calf  and  sheep,  especially  in  an  infusion  of  the 
fundus  part.  In  other  mammals  and  in  birds  it  is  seldom  found,  and  in 
fishes  hardly  ever  in  the  neutral  infusion.  In  these  cases,  as  in  man  and 
the  higher  animals,  a  rennin-forming  substance,  a  rennin  zymogen,  occurs, 
which  is  converted  into  rennin  by  the  action  of  an  acid  (Hammarstex). 
We  have  no  knowledge  as  to  whether  the  rennet  enzyme  found  in  different 
animals  is  chymosin  or  parachymosin.  Enzymes  acting  like  rennin  are 
also  found  in  the  blood  and  several  organs  of  higher  animals,  as  well  as  in 
invertebrates.  Similar  enzymes  also  occur  widely  diffused  in  the  plant 
kingdom,  and  numerous  micro-organisms  have  the  power  of  producing 
rennin  enzymes.  Also  antibodies  to  the  rennet  enzymes,  atitichymosins, 
occur  in  the  animal  kingdom,  as  shown  by  Hamm.arstex  and  Rodex  in 
blood-serum,  and  ma}-  be  produced  in  the  animal  body  by  the  introduction 
of  rennin  into  the  latter  (Morcexroth^). 

'  Deutsch.  med.  Wochenschr  ,  1899.  and  Pfliiger's  .\rch.,  79. 

^  Schumbiug,  Virchow's  Arch.,  97,  A  good  review  of  the  literature  may  be  foimd 
in  Szydlowski,  Beitrage  zur  Kenntnis  des  LabenzjTn  nach  Beobachtungen  an  Saug- 
lingen,  Jahrb.  f.  Kinderheilkunde  (N.  F.),  S-l.  See  also  Lorcher,  Pfliiger's  Arch.,  69, 
which  also  contains  the  pertinent  literature.  An  excellent  review  of  the  literature  on 
rennin  and  its  action  may  be  found  in  E.  Fuld.  Ergebnisse  der  Physiol  .  1,  Abt.  1,  468. 

^  See  Roden,  Upsala  Lakaref.  Forh.,-  22;  Morgenroth,    Centralbl.    f.    Bakter.,   26 


362  DIGESTION. 

Rennin  is  just  as  difficult  to  prepare  in  a  pure  state  as  the  other  enzymes. 
The  purest  rennin  enzyme  thus  far  obtained  did  not  give  the  ordinary 
protein  reactions.  On  heating  its  solution  rennin  is  more  or  less  quickly 
destroyed,  depending  upon  the  length  of  heating  and  upon  the  concentra- 
tion. If  an  active  and  strong  infusion  of  the  gastric  mucosa  in  water 
containing  3  p.  m.  HCl  is  heated  to  37-40°  C.  for  48  hours,  the  rennin  is 
destroyed,  while  the  pepsin  remains.  A  pepsin  solution  free  from  rennin 
can  be  obtained  in  this  way.  Rennin  is  characterized  by  its  physiological 
action,  which  consists  in  coagulating  milk  or  a  casein  solution  containing 
lime,  if  neutral  or  very  faintly  alkaline.  The  law  of  the  action  of  this 
■enzyme  is  different  from  that  of  the  action  of  pepsin.  As  specially  shown  by 
FuLD,  within  certain  limits,  the  coagulation  time,  T,  is  equal  to  a  con- 
stant, C,  divided  by  the  quantity  of  rennin,  L.  As  shown  by  Bang,^ 
this  law  does  not  apply  to  parachymosin. 

From  the  different  laws  of  pepsin  and  rennin  action  it  follows  that  the  repeated 
appearance  recently  of  the  view  of  Pawlow's  school,  that  pepsin  and  rennin  are 
the  same  bodies,  cannot  be  correct.  The  experiments  given  by  Pawlow  and 
Parastschuk  as  proof  for  this  view  are  unfortunately  in  error  in  principle.  Also 
the  investigations  published  by  Sawjalow  are  not  decisive,  and  the  recent  work 
of  Schmidt-Nielsen  ^  has  given  new  proofs  for  the  non-identity  of  the  two  en- 
zymes. According  to  Nencki  and  Sieber,-  with  whom  Pekelharing  agrees, 
the  enzyme  of  the  gastric  juice  forms  a  gigantic  molecule  which  is  able  to  perform 
the  different  actions  at  the  same  time,  although  each  enzyme  action  is  connected 
with  a  certain  atomic  complex.  Such  a  view  might  appear  plausible,  especially  as 
pepsin  and  rennin  enzyme  regularly  occur  together  in  the  animal  kingdom.  As 
the  body  which  is  precipitated  from  gastric  juice  by  cooling  and  which  forms 
the  ferment  has  been  shown  to  be  a  mixture,  and  also  as  the  two  enzymes  and 
also  the  proenzymes  have  been  separated  by  Glaessner  ^  from  each  other,  this 
view  does  not  seem  to  be  sufficiently  well  grounded. 

Parachymosin  differs  from  chymosin  by  being  much  more  resistant 
towards  acids,  but  is  more  readily  destroyed  by  alkalies.  Calcium  chloride 
accelerates  the  casein  coagulation  with  parachymosin  very  much  more 
than  with  chymosin. 

Rennin,  like  other  enzymes,  may  be  carried  down  by  other  precipitates 
and  thus  may  be  obtained  relatively  pure.  It  may  also  be  obtained,  con- 
taminated with  a  great  deal  of  proteins,  by  extracting  the  mucous  coat  of 
the  stomach  with  glycerine. 

A  comparatively  pure  solution  of  rennin  may  be  obtained  in  the  follow- 
ing way.  An  infusion  of  the  mucous  coat  of  the  stomach  in  hydrochloric 
acid  is  prepared  and  then  neutralized,  after  which  it  is  repeatedly  shaken 
with  new  quantities  of  magnesium  carbonate  until  the  pepsin  is  precipi- 


•  Fuld,  Hofmeister's  lieitriige,  2;   Bang,  1.  c. 

^  Pawlow  and  Parastschuk,  Zeitschr.  f.  physiol.  Chem.,  42;  Sawjalow,  ibid.,  46; 
Schmidt-Nielsen,  ibid.,  48. 

3  Glaessner,  Hofmeister's  Beitrage,  1;  Nencki  and  Sieber,  Zeitschr.  f.  physiol. 
Chem.,  32;  Pekelliaring,  foot-notes,  and  3,  p.  355. 


GASTRIC   LIPASE.  303 

tated.  The  filtrate,  which  sliould  act  strongly  on  milk,  is  precipitated  by 
basic  lead  acetate,  the  precipitate  decomposed  with  very  dilute  sulphuric 
acid,  the  acid  liquid  filtered  and  treated  with  a  solution  of  stearin  soap. 
The  rennin  is  carried  down  by  the  fatty  acids  set  free,  and  when  these  last 
are  placed  in  water  and  removed  by  shaking  with  ether,  the  rennin  remains 
in  the  watery  solution. 

Plastein.  As  mentioned  on  page  56,  Danilewsky  first  showed  the 
power  of  rennin  solutions  of  causing  a  partial  coagulation  of  proteoses  and 
of  converting  them  into  so-called  plastein.  This  action,  which  is  also 
ascribed  to  other  enzyme  solutions  (see  page  56),  has  probably  nothing  to 
do  with  the  rennin  enzyme,  but  depends  more  likely  upon  another  enzyme. 
The  nature  of  these  enzymes,  as  well  as  the  manner  and  importance  of  the 
plastein  formation,  is  still  unknown. 

Gastric  Lipase  (stomach  steapsin).  F.  Volhard  ^  has  made  the  dis- 
covery that  the  gastric  juice  has  a  strong  fat-splitting  action  only  when 
the  fat  is  in  a  fine  emulsion,  as  in  the  yolk  of  the  egg,  in  milk  or  in  cream. 
This  action,  which  is  still  not  undisputed  (Inouye),  depends  upon  an  enzyme 
which  can  be  extracted  from  the  mucosa  by  glycerine,  and  whose  action, 
it  seems,  follows  SchIjtz's  law  for  pepsin,  that  the  quantity  is  proportional 
to  the  square  of  the  quantity  of  enzymotic  products.  This  enzyme,  which 
seems  to  be  produced  from  a  zymogen,  does  not  behave  in  the  same  way 
W'hen  obtained  from  different  animals.  The  enzyme  from  the  pig  stomach 
is  very  sensitive  towards  acids  but  less  sensitive  towards  alkalies.  That 
from  the  dog  and  from  the  human  stomach  is,  on  the  contrary,  sensitive 
tow^ards  alkalies,  while  comparatively  resistant  towards  acids  (Fromme). 
The  enzyme  is  only  slowly  extracted  from  the  pig  stomach  by  glycerine,  and 
the  extract  shows  its  maximal  activity  only  after  several  days.  The  filtered 
glycerine  extract  is  inactive  (Fromme).  Falloise,  by  experiments  on 
rabbits  and  dogs,  has  shown  that  gastric  lipase  is  not  derived  by  regurgita- 
tion from  the  intestine,  nor  does  it  come  from  the  pancreas  by  means  of 
the  blood,  but  it  is  formed  in  the  stomach.  Laqueur,  by  means  of 
experiments  with  the  filtered  juice  obtained  from  a  small  Pawlow  stomach, 
has  shown  that  this  juice  had  a  splitting  action  upon  yolk  emulsion.  Gas- 
tric lipase,  according  to  Laqueur,-  cannot  be  pancreas  steapsin,  and  is 
also  not  an  intracellular  tissue  enzyme,  but  is  secreted  by  the  glands. 

The  question  whether  the  parietal  cells  principally  or  the  chief  cells, 
or  both,  take  part  in  the  formation  of  free  acid  is  somewhat  disputed.^ 

*  Volhard,  Miinch.  med.  Wochenschr.,  1900,  and  Zeitschr.  f.  klin.  Med.,  42,  43. 
See  also  Stade,  Hofmeister's  Beitriige,  3;  A.  Fromme,  ibid.,  7;  A.  Zins._er,  ibid.;  H. 
Engel,  ibid.;  and  Inouye,  Arch.  f.  Verdauungskrank.,  9. 

^  Falloise,  Arch.  int.  de  Physiol.,  3  (1906);   Laqueur,  Hofmeister's  Beitrage,  8. 

^  See  Heidenhain,  Pfliiger's  Arch.,  IS  and  19,  and  Hermann's  Handbuch,  5,  part  I, 
"Absonderungsvorgange";   Klemensiewicz,  Wien.  Sitzungsber.,  71;  Frankel,  Pfliiger'a 


364  DIGESTION. 

There  can  be  no  doubt  that  the  hydrochloric  acid  of  the  gastric  juice 
originates  from  the  chlorides  of  the  blood,  because,  as  is  well  known,  a 
secretion  of  perfectly  typical  gastric  juice  takes  place  in  the  stomachs  of 
fasting  or  starving  animals.  As  the  chlorides  of  the  blood  are  derived 
from  the  food,  it  is  easily  understood,  as  shown  by  Cahn,^  that  m  dogs 
after  a  sufficiently  long  common-salt  starvation  the  stomach  secreted  a 
gastric  juice  containing  pepsin,  but  no  free  hydrochloric  acid.  On  the 
administration  of  soluble  chlorides,  a  gastric  juice  containing  hydrochloric 
acid  was  immediately  secreted.  On  the  introduction  of  alkali  iodides  or 
bromides,  KtJLz,  Nencki  and  Schoumow-Simanowski  ^  have  shown  that 
the  hydrochloric  acid  of  the  gastric  juice  is  replaced  by  HBr,  and  to  a 
less  extent  by  HI.  According  to  Koeppe  the  seat  of  formation  of  hydro- 
chloric acid  is  not  the  blood  nor  the  glands,  but  the  interior  of  the  stomach 
in  the  immediate  neighborhood  of  the  w^all.  The  hydrochloric  acid  is 
assumed  to  be  produced  from  the  chlorides  of  the  food,  as  the  semiper- 
meable wall  is  not  permeable  for  the  CI  ions  but  is  for  the  Na  ions  and  for 
the  H  ions.  As  the  Na  ions  leave  the  stomach  contents  an  equivalent 
quantity  of  H  ions  wander  from  the  blood  through  the  stomach  wall  into 
the  interior  of  the  stomach  and  combine  with  the  CI  ions.  This  theory  is 
difficult  to  reconcile  with  the  fact  that  in  dogs  with  apparent  feeding  the 
empty  stomach  secretes  abmidant  juice.  Other  objections  have  also  been 
raised  against  this  by  Beneath  and  Sachs.  The  secretion  of  free  hydro- 
chloric acid  from  the  alkaline  blood  has  been  explained  in  other  ways 
(Maly,  Bunge,  L.  Schwarz),  but  as  yet  no  satisfactory  theory  has  been 
suggested.^ 

After  a  full  meal,  when  the  store  of  pepsin  in  the  stomach  is  completely 
exhausted,  Schiff  claims  that  certain  bodies,  especially  dextrin,  have 
the  property  of  causing  a  supply  of  pepsin  in  the  mucous  membrane.  This 
"charge  theory,"  though  experimentally  tested  by  several  investigators, 
has  nevertheless  not  yet  been  confirmed.  On  the  contrary,  the  state- 
ment of  Schiff  that  a  substance  forming  pepsin,  a  "pepsinogen"  or  "pro- 
pepsin," occurs  in  the  ventricle  has  been  proved.  Langley  *  has  shown 
positively  the  existence  of  such  a  sulistance  in  the  mucous  coat.     This 

Arch.,  48  and  50;  Contejean,  1.  c,  Chapter  II;  Kranenburg,  Archives  Teyler,  Series 
II,  Haarlem,  1901,  and  Mosse,  Centralbl.  f.  Physiol.,  17,  217. 

'  Zeitschr.  f.  physiol.  Chem.,  10. 

2  Kulz,  Zeitschr.  f.  Biologic,  23;  Nencki  and  Schoumow,  Arch,  des  sciences  bid. 
de  St.  P^tersbourg,  3. 

'  Koeppe,  Pfliiger's  Arch.,  62;  Benrath  and  Sachs,  ibid.,  109;  Maly,  see  v.  Bunge's 
Lehrbuch  der  physiol.  u.  pathol.  Chem.,  4.  Aufl.,  1898;  Schwarz,  Hofmeister's  Bei- 
triige,  5. 

*  Schiff ,  Letjons  sur  la  physiol.  de  la  digfotion,  18G7,  2;  Langley  and  Edkins, 
Journ.  of  Physiol.,  7. 


PYLORIC  SECRETION.  365 

substance,  propepsin,  shows  a  comparatively  strong  resistance  to  dilute 
alkalies  (a  soda  solution  of  5  p.  m.),  which  easily  destroy  pepsin  (Langley). 
Pepsin,  on  the  other  hand,  withstands  better  than  propepsin  the  action  of 
carbon  dioxide,  which  quickly  destroys  the  latter.  The  occurrence  of  a 
remiin  zymogen,  and  possibly  also  of  a  steapsmogen,  m  the  mucous  coat 
has  been  mentioned  above. 

According  to  Herzen  and  his  collaborators  *  we  must  differentiate  between 
pepsinogens  and  bodies  accelerating  the  flow  of  juice.  To  the  first  belong  inulin 
and  glycogen,  while  alcohol  belongs  to  the  latter  class  of  bodies.  Dextrin  not 
only  accelerates  the  flow  of  juice,  but  also  acts  as  a  pepsinogen,  especially  as  the 
latter.  Meat  extract  which  has  both  actions  is  especially  a  flow-accelerator. 
The  pepsinogen  action  consists  in  converting  the  zymogen  into  pepsin  and  in 
this  way  increases  the  quantity  of  pepsin  ;  the  flow-accelerating  substances  in- 
crease the  quantity  of  secreted  juice. 

The  question  in  what  cells  the  two  zymogens,  especially  the  propepsin, 
are  produced  has  been  extensively  discussed  for  several  years.  Formerly  it 
was  the  general  opinion  that  the  parietal  cells  were  pepsin  cells,  but  since 
the  investigations  of  Heidenhain  and  his  pupils,  Laxgley  and  others,  the 
formation  of  pepsin  has  been  attributed  to  the  chief  cells.^ 

The  Pyloric  Secretion.  That  part  of  the  pyloric  end  of  the  dog's 
stomach  which  contains  no  fundus  glands  was  dissected  by  Klemexsie- 
wicz,  one  end  being  sewed  together  in  the  shape  of  a  blind  sac  and  the 
other  sewed  into  the  stomach.  From  the  fistula  thus  created  he  was  able 
to  obtain  the  pyloric  secretion  of  a  living  animal.  This  secretion  is  alka- 
line, viscous,  jelly-like,  rich  in  mucin,  of  a  specific  gravity  of  1.009-1.010, 
and  containing  16.5-20.5  p.  m.  solids.  It  habitually  contains  pepsin, 
which  has  been  proved  by  Heidenhain  by  observations  on  a  permanent 
pyloric  fistula,  and  the  amount  may  sometimes  be  considerable.  Conte- 
jean  has  investigated  the  pyloric  secretion  in  other  ways,  and  finds  that 
it  contains  both  acid  and  pepsin.  The  alkaline  reaction  of  the  secretions 
investigated  by  Heidenhain  and  Klemensieavicz  is  due,  according  to 
CoNTEjEAN,  to  an  abnormal  secretion  caused  by  the  operation,  because 
the  stomach  readily  yields  an  alkaline  juice  instead  of  an  acid  one  under 
abnormal  conditions.  The  statements  of  Heidenhain  and  Klemensie- 
wicz  have  been  substantiated  by  Akermann,  while  Kresteff,  w^ho 
operated  according  to  another  method,  and  Schemiakine  ^  have  come 
to  the  same  results.  Kresteff  found  in  the  juice  (of  dogs)  pepsin,  but  no 
chymosin.     Yerhaegen  ^  has  observed  in  hiunan  beings  towards  the  end 

'  Pfliiger's  Arch.,  84. 

^  See  foot-note  3,  p.  364. 

^Heidenhain  and  IClemensiewicz,  1.  c. ;  Contejean,  I.  c,  Chapter  11,  and  Skand. 
Arch.  f.  Physiol.,  6;  Akermann,  ibid.,  5;  Ivresteff,  Maly's  Jaliresber.,  30;  Schemia- 
kine, Arch,  des  scienc.  biolog.  de  St.  Petersboiirg,  10. 

*  See  the  work  of  Verhaegen,  "La  Cellule,"  1896,  1897. 


366  DIGESTION. 

of  the  ventricle  digestion  a  non-fluid  acid,  which,  according  to  him,  originates 
in  the  pyloric  region. 

The  secretion  of  gastric  juice  under  different  conditions  may  x&iry  con- 
siderably. The  statements  concerning  the  quantity  of  gastric  juice  secreted 
in  a  certain  time  are  therefore  so  unreliable  that  they  need  not  be  taken 
into  account. 

The  Chyme  and  the  Digestion  in  the  Stomach.  By  means  of  the  chem- 
ical stimulation  caused  by  the  food,  a  copious  secretion  of  gastric  juice 
occurs,  which  gradually  mixes  with  the  swallowed  food,  and  digests  it 
more  or  less  strongly.  The  material  in  the  stomach  during  digestion 
which  has  a  pasty  or  thick  consistency  and  is  called  chyme,  is  not  a 
homogeneous  mixture  of  the  ingesta  with  the  various  digestive  fluids, 
gastric  juice,  saliva,  and  gastric  mucus,  but  the  conditions  seem  to  be 
more  complicated. 

From  the  investigations  of  several  workers,  such  as  Hofmeister  and 
ScHuTZ,  MoRiTZ,  Caxxox,  Schemiakixe.^  and  others,  on  the  movements 
of  the  stomach,  we  conclude  that  this  organ  consists  of  two  phj-siologically 
different  parts,  the  pylorus  and  the  fundus.  The  greater  fundus  part, 
which  serves  essentially  as  a  reservoir,  may  by  a  rhythmic,  strong  con- 
traction of  the  muscle,  acting  like  a  sphincter  between  it  and  the  pylorus 
part,  be  separated  from  the  latter,  and  according  to  some  observers  sa 
completely  so  that  during  contraction  almost  nothing  passes  from  the 
fundus  to  the  pylorus  part.  Differing  from  the  fundus  part,  the  pylorus 
is  the  seat  of  ver}-  powerful  contractions  by  which  its  contents  are  intimately 
mixed  with  gastric  juice  and  are  also  driven  through  the  pyloric  valve  into 
the  intestine. 

The  contents  of  the  pylorus  part  have  an  acid  reaction,  and  a  strong 
pepsin  digestion  takes  place  in  the  contents,  which  are  thoroughly  mixed 
with  gastric  juice.  By  ver}-  instructive  investigations  on  different  animals 
(frogs,  rats,  rabbits,  guinea-pigs,  and  dogs)  Grutzxer^  has  shown,  when 
the  animals  are  fed  with  food  having  different  colors,  and  the  stomach 
removed  after  a  certain  time,  and  the  contents  frozen,  that  the  frozen, 
sections  show  a  regular  stratification  of  the  contents.  These  layers  are 
so  arranged  that  the  food  first  taken  is  found  in  direct  contact  with  the 
mucosa,  while  the  food  taken  later  is  inclosed  by  that  partaken  of  first, 
and  this  prevents  contact  with  the  walls  of  the  stomach.  The  empty 
stomach,  whose  walls  touch  each  other,  is  so  filled  that,  as  a  rule,  the 
foodstuffs  taken  later  are  in  the  middle  of  the  older  food. 

Because  of  this  fact  onlv  the  foodstuffs  which  lie  close  to  the  surface  of 


*  Hofmeister  and  Schiitz,  Arch.  f.  exp.  Path.  u.    Pharm.,  20;  Moritz,  Zeitschr.  f. 
Biologie,  32;  Cannon,  Amer.  Journ.  of  Physiol.,  1;   Schemiakine,  1.  c. 
» Pfliiger's  Arch.,  106. 


DIGESTION    IN    THE   STO^kUCH.  367 

the  mucous  membrane  undergo  the  process  of  peptic  digestion,  and  it  is 
principally  this  ingesta,  which  lies  on  the  surface  and  is  laden  with  pepsin  and 
mixed  with  gastric  juice,  which  is  shoved  to  the  pylorus  end,  here  mixed 
and  digested,  and  finally  moved  into  the  intestine.  The  fundus  part  is 
therefore  less  a  digestion-organ  than  a  storage-organ,  and  in  the  interior 
of  the  same  the  food  may  remain  for  hours  without  coming  in  contact 
with  a  trace  of  gastric  juice. 

What  has  been  said  above  applies  at  least  to  solid  food.  We  have  no- 
extensive  oljservations  on  the  behavior  of  fluids  or  semifluid  food.  Accord- 
ing to  Grutzxer,  also  in  these  cases,  as  well  as  in  the  above-mentioned 
experiments,  the  swallowed  foodstuffs  are  not  irregularly  mixed  together. 
Fluids  quickly  leave  the  stomach,  which  is  also  the  case  with  a  mixture  of 
solid  and  fluid  food. 

The  fact  that  only  that  part  of  the  ingesta  lying  on  the  mucous  mem- 
brane is  mixed  with  gastric  juice,  while  the  mass  in  the  interior  is  not  acid 
in  reaction,  is  of  special  importance  for  the  digestion  of  starches  in  the 
stomach.  By  this  we  can  explain  why  the  salivar}-  diastase,  although 
sensitive  towards  acids,  can  continue  its  action  for  a  long  time  in  the  con- 
tents of  the  stomach.  Caxxox  and  Day  ^  have  shown  that  this  is  true 
by  special  experiments  upon  animals,  and  the  occurrence  of  sugar  and 
dextrin  in  the  contents  of  the  human  stomach  has  been  repeatedh-  observ'ed. 
In  camivora,  whose  saliva  shows  nearly  no  diastatic  action,  it  is  a  priori 
not  expected  that  there  should  be  a  diastatic  action  in  the  stomach — of 
course  excluding  the  action  of  micro-organisms  which  may  be  present. 
Still,  according  to  Friedexthal.  dogs  can  readily  digest  starch,  and  the 
gastric  juice  of  dogs,  according  to  him,  contains  a  diastatic  enzyme  which 
is  active  even  in  strong  acid  reaction. - 

The  gastric  contents  which  have  been  prepared  in  the  pylorus  part  are 
passed  through  the  pylorus  into  the  intestine  intermittently.  This  mate- 
rial is  generally  fluid,  but  it  is  possible  that  pieces  of  solid  food  ma}-  also 
occur,  and  this  has  been  often  observed.  From  Busch's  observations  on  a 
human  intestinal  fistula,  it  required  generally  15-30  minutes  after  eating 
for  undigested  food  to  pass  into  the  upper  part  of  the  small  intestine.  In 
a  case  of  duodenal  fistula  in  a  human  being  observed  by  Kuhxe,  he  saw, 
ten  minutes  after  eating,  uncurdled  but  still  coagulable  milk  and  small 
pieces  of  meat  pass  out  of  the  fistula.  The  time  in  which  the  stomach 
unburdens  itself  of  its  contents  depends  naturally  upon  the  coarseness  or 
fineness  of  the  food;  essentially,  however,  it  depends  upon  the  reflex  action 


'Cannon  and  Day,  Amer.  Journ.  of  Physiol.,  9;  Friedenthal,  Arch.  f.  (.\nat.  u.) 
Physiol.,  1899,  .>^uppl. 

*  See  Scheunert  and  Grimmer.  Zeitschr.  f.  physiol.  Chem.,  48,  on  the  occurrence  of 
dias'atic  enzjTnes  in  plant  foodstuffs. 


368  DIGESTION. 

from  the  stomach  or  intestine  causing  an  opening  or  closing  of  the  pylorus, 
which  action  is  dependent  upon  the  quantity  and  character  of  the  food, 
the  amount  of  fat,  and  the  degree  of  acidity  in  the  contents  of  the 
stomach  and  intestine.  The  emptying  of  the  food  into  the  small 
intestine  causes,  as  shown  by  Pawlow,  a  closing  of  the  pylorus  by  chemo- 
reflex  in  which  the  hydrochloric  acid  and  the  fat  take  part,  and  we  thus 
find  in  this  regard  an  alternate  action  between  the  stomach  and  duodenum. 
The  time  necessary  for  the  stomach  to  empty  itself  must  differ  considerably 
under  various  conditions.  Beaumont  ^  found  in  his  extensive  observations 
on  the  Canadian  hunter  St.  Martin  that  the  stomach,  as  a  rule,  is  emptied 
1^-5|  hours  after  a  meal,  depending  upon  the  character  of  the  food. 

The  time  in  which  different  foods  leave  the  stomach  depends  also  upon 
their  digestibility.  In  regard  to  the  unequal  digestibility  in  the  stomach 
of  foods  rich  in  proteins,  which  really  form  the  object  of  the  action  of  the 
gastric  juice,  a  distinction  must  be  made  between  the  rapidity  with  which 
the  proteins  are  converted  into  proteoses  and  peptones  and  the  rapidity 
with  which  the  food  is  converted  into  chyme,  or  at  least  so  prepared  that 
it  may  easily  pass  into  the  intestine.  This  distinction  is  especially  im- 
portant from  a  practical  standpoint.  When  a  proper  food  is  to  be  decided 
upon  in  cases  of  diminished  gastric  digestion,  it  is  important  to  select  such 
foods  as  leave  the  stomach  easily  and  quickly,  independently  of  the  diffi- 
culty or  ease  with  which  their  protein  is  peptonized,  and  which  require  as 
little  action  as  possible  on  the  part  of  this  organ.  From  this  point  of  view 
those  foods,  as  a  rule,  are  most  digestible  which  are  fluid  from  the  start  or 
may  be  easily  liquefied  in  the  stomach;  but  these  foods  are  not  always 
the  most  digestible  in  the  sense  that  their  protein  is  most  easily  peptonized. 
As  an  example,  hard-boiled  white  of  egg  is  more  easily  peptonized  than 
fluid  white  of  egg  at  a  degree  of  acidity  of  1-2  p.  m.  HCl;^  nevertheless 
we  consider,  and  justly,  that  an  unboiled  or  soft-boiled  egg  is  easier  to 
digest  than  a  hard-boiled  one.  Likewise  uncooked  meat,  when  it  is  not 
chopped  very  fine,  is  not  more  quickly  but  more  slowly  peptonized  by  the 
gastric  juice  than  the  cooked,  but  if  it  is  divided  sufficiently  fine  it  is  often 
more  quickly  peptonized  than  the  cooked. 

The  greater  or  less  facility  with  which  the  different  protein  foods  are 
digested  in  the  stomach  has  been  comparatively  little  studied.  The  most 
complete  investigations  on  this  subject  are  those  of  Fermi,^  but  as  they 
do  not  allow  of  a  short  discussion  we  must  refer  to  the  original  publication. 

*  Busch,  Virchow's  Arch.,  14;  Kiihne,  Lehrb.  d.  physiol.  Chem.,  53;  (Pawlow  and) 
Serdjukow,  Maly's  Jahresber.,  29;  Lintwarew,  Biochem.  Centralbl.,  1,  96.  See  also 
Richet,  1.  c;  Beaumont,  The  Physiology  of  Digestion,  1833. 

^  Wawrinsky,  Maly's  Jahresber.,  3. 

'Arch.  f.  (Anat.  u.)  Physiol.,  1901,  Suppl. 


DIGESTION    IN   THE   STOMACH.  369 

The  rapidity  with  which  different  foods  leave  the  stomach  has  been 
studied  by  Cannon  ^  in  the  case  of  cats.  After  preliminary  hunger  the  animals 
received  different  food,  such  as  meat,  fat,  and  carbohydrate  mixed  with 
bismuth  subnitrate,  and  then  with  the  aid  of  the  Rontgen  rays  the  time 
was  noted  when  the  food  passed  into  the  intestine.  The  carbohydrate  leaves 
the  stomach  first,  the  proteins  next,  and  the  fats  last.  If  the  carbohy- 
drate is  given  before  the  protein  food,  then  it  leaves  the  stomach  with 
ordinary  rapidity;  while  if  protein  food  and  then  carbohydrate  is  given 
the  passage  of  the  carbohydrate  is  retarded.  A  mixture  of  protein  food  and 
carbohydrates  leaves  the  stomach  more  slowly  than  carbohydrates  alone,  but 
faster  than  protein  food  alone.  The  fat,  which  remains  in  the  stomach 
for  a  long  time  and  leaves  the  stomach  only  in  amounts  which  are  absorbed 
or  removed  from  the  duodenum,  retards  the  passage  of  the  protein  foods 
as  well  as  the  carbohydrates. 

As  our  knowledge  of  the  digestibility  of  the  different  foods  in  the 
stomach  is  slight  and  dubious,  so  also  our  knowledge  of  the  action  of  other 
bodies,  such  as  alcoholic  drinks,  bitter  principles,  spices,  etc.,  on  the  nat- 
ural digestion  is  very  uncertain  and  imperfect.  The  difficulties  which 
stand  in  the  way  of  this  kind  of  investigation  are  very  great,  and  there- 
fore the  results  obtained  thus  far  are  often  ambiguous  or  conflict  with 
each  other.  For  example,  certain  investigators  have  observed  that  small 
quantities  of  alcohol  or  alcoholic  drinks  do  not  prevent  but  rather  facili- 
tate digestion;  others  observe  only  a  disturbing  action,  while  other  investi- 
gators report  having  found  that  the  alcohol  first  acts  somewhat  as  a 
disturbing  agent,  but  afterwards,  when  it  is  absorbed,  produces  an  abund- 
ant secretion  of  gastric  juice,  and  thereby  facilitates  digestion  (Gluzinski, 
Chittenden  ^).  The  accelerating  action  of  alcohol  upon  the  flow  of  gastric 
juice  has  already  been  mentioned  on  page  352. 

The  digestion  of  sundry  foods  is  not  dependent  on  one  organ  alone,  but 
is  divided  among  several.  For  this  reason  it  is  to  be  expected  that  the 
various  digestive  organs  can  act  for  one  another  to  a  certain  point,  and 
that  therefore  the  work  of  the  stomach  could  be  taken  up  more  or  less 
by  the  intestine.  This  in  fact  is  the  case.  Thus  the  stomachs  of  dogs 
and  cats  have  been  completely  extirpated  or  nearly  so  (Czerny,  Carvallo 
and  Pachon),  and  also  that  part  necessary  in  the  digestive  process  has 
been  eliminated  by  plugging  the  pyloric  opening  (Ludwig  and  Ogata), 
and  in  both  cases  it  was  possible  to  keep  the  animal  alive,  well  fed,  and 
strong  for  a  shorter  or  longer  time.     This  is  also  true  for  human  being.=.3 

^  Amer.  Journ.  of  Physiol.,  12. 

^  Gluzinski,  Deutsch.  Arch.  f.  klin.  Med.,  39;  Chittenden,  Centralbl.  f.  d.  med.  Wis- 
eensch.,  1889;   Chittenden  and  Mendel  and  others,  Amer.  Journ.  of  Physiol.,  1. 

^Czerny,  cited  from  Bunge,  Lehrbuch  d.  physiol.  u.  path.  Chem.  4.  Aufl.,  Theil  2, 
173;     Carvallo    and   Pachon,    Arch.  d.  Physiol.   (5),  7;    Ogata,    Arch.   f.  (Anat.  u.) 


370  DIGESTION. 

In  these  cases  it  is  evident  that  the  digestive  work  of  the  stomach  was 
taken  up  by  the  intestine;  but  all  food  cannot  be  digested  in  these  cases 
to  the  same  extent,  and  the  connective  tissue  of  meat  especially  is  some- 
times found  to  a  considerable  extent  undigested  in  the  excrements. 

In  order  to  judge  of  the  role  of  the  stomach  in  digestion  the  amount  of 
the  digestion  products  in  the  stomach  has  been  determined.  These  deter- 
minations, partly  on  man  and  partly  on  animals,  have  led,  as  is  to  be 
expected,  to  varying  results  (Cahn,  Ellenberger  and  Hofmeister, 
Chittenden  and  Amerman).  The  recent  investigations  of  E.  Zunz  show 
that  boiled  meat  in  the  stomach  of  a  dog  yields  chiefly  proteoses  with  small 
amounts  of  simpler  cleavage  products,  and  only  very  little  acid  albumin  is 
formed.  The  quantity  of  proteose  nitrogen  was  90  per  cent  of  the  nitrogen 
of  the  non-coagulable  substances.  Tobler,  who  has  studied  in  dogs  with 
duodenal  fistula,  the  digestion  in  the  stomach  under  conditions  which 
were  as  nearly  physiologically  normal  as  possible  has  arrived  at  different 
results.  After  feeding  finely  chopped  meat  freed  from  extractives  by  water, 
he  found  that  about  20  to  30  per  cent  of  the  total  nitrogen  was  absorbed  in 
the  stomach,  while  about  20  per  cent  left  the  stomach  in  an  insoluble  form 
and  50  to  65  per  cent  in  solution.  The  chief  mass  of  the  dissolved  protein 
(about  80  per  cent)  consisted  of  peptones,  the  remainder  of  proteoses. 
London  and  Sulima  ^  have  arrived  at  different  results.  They  experi- 
mented upon  dogs,  making  various  fistulse,  such  as  gastric,  pyloric,  and 
duodenal  fistulse,  and  used  as  food  partly  hard-boiled  egg,  which  was  given 
to  the  dogs  in  quantities  of  200  grams  and  in  as  large  pieces  as  possible,  and 
partly  raw  white  of  egg.  In  both  cases,  contrary  to  the  experience  of 
Tobler,  the  proteins  were  not  absorbed  in  the  stomach.  They  also  found 
in  the  dog  with  pyloric  fistula  that  after  feeding  200  grams  boiled  protein 
the  chief  portion  of  the  digestion  products  (about  87  per  cent)  left  the 
stomach  within  the  first  two  hours  of  digestion.  Of  the  cleavage  prod- 
ucts the  contents  of  the  stomach  contained  chiefly  proteoses,  while  in 
the  material  which  left  the  stomach  in  the  dog  with  pyloric  fistula,  the 
peptones  prevailed.  The  proteoses  seem  to  be  retained  for  a  longer  time  in 
the  stomach,  making  a  further  transformation  into  peptones  possible. 

As  is  to  be  expected,  the  behavior  is  not  always  the  same,  and  important 
variations  often  occur.  It  is,  however,  quite  generally  assumed  that  no 
peptonization  of  the  proteins  worth  mentioning  occurs  in  the  stomach, 

Physiol.,  1883;  Groh^,  Arch.  f.  exp.  Path.  u.  Pharm.,  49.  In  regard  to  a  human 
case  of  Schlatter  see  Wroblewski,  Centralbl.  f.  Physiol.,  11,  665. 

'  Cahn,  Zeitschr.  f.  klin.  Med.,  12;  Ellenberger  and  Hofmeister,  Arch.  f.  (Anat.  u.) 
Phyisol.,  1S90;  Chittenden  and  Amerman,  Journ.  of  Physiol.,  14;  E.  Zunz,  Hofmeister's 
Beitrage,  3.  See  Reach,  ibid.,  4.  See  also  Zunz,  Annal.  de  la  soc.  roy.  d.  scienc.  mdd. 
et  natur.  de  Bruxelles,  12  and  13;  Tobler,  Zeitsclu".  f.  physiol.  Chem.,  45;  London, 
and  Sulima,  ibid.,  4fi. 


DIGESTION   IX    THE  STOMACH.  371 

and  that  the  protein  foods  are  only  prepared  in  the  stomach  for  the  real 
digestive  processes  in  the  intestine.  That  the  stomach,  at  least  the  fundus 
part,  serves  in  the  first  place  as  a  storeroom  follows  from  its  shape,  and 
this  function  is  of  special  value  in  certain  new-bom  animals,  for  instance 
in  dogs  and  cats.  In  these  animals  the  secretion  of  the  stomach  contains 
only  hydrochloric  acid  but  no  pepsin,  and  the  casein  of  the  milk  is  converted 
by  the  acid  alone  into  solid  lumps  or  a  solid  coagulum  which  fills  the  stomach. 
Small  portions  of  this  coagulum  pass  into  the  intestine  only  little  by  little, 
and  an  overburdening  of  the  intestine  is  thus  prevented.  In  other  animals, 
such  as  the  snake  and  certain  fishes  which  swallow  their  food  entire,  it  is 
certain  that  the  major  part  of  the  process  of  digestion  takes  place  in  the 
stomach.  The  importance  of  the  stomach  in  digestion  cannot  at  once  be 
decided.  It  varies  for  different  animals,  and  it  may  vary  in  the  same 
animal,  depending  upon  the  division  of  the  food,  the  rapidity  with  which 
the  peptonization  takes  place,  the  more  or  less  rapid  increase  in  the  amount 
of  hydrochloric  acid,  and  so  on. 

It  is  a  well-known  fact  that  the  contents  of  the  stomach  may  be  kept 
without  decomposing  for  some  time  by  means  of  hydrochloric  acid,  while, 
on  the  contrary',  when  the  acid  is  neutralized  a  fermentation  commences 
by  w^hich  lactic  acid  and  other  organic  acids  are  formed.  According  to 
CoHX  an  amount  of  hydrochloric  acid  more  than  0.7  p.  m.  completely 
arrests  lactic-acid  fermentation,  even  under  othenvise  favorable  circum- 
stances, and  according  to  Strauss  and  Bialocour  the  limit  of  lactic-acid 
fermentation  lies  at  1.2  p.  m.  hydrochloric  acid  united  to  organic  bodies. 
The  hydrochloric  acid  of  the  gastric  juice  has  unquestionably  an  antifer- 
mentative  action,  and  also,  like  all  dilute  mineral  acids,  an  antiseptic  action. 
This  action  is  of  importance,  as  many  pathogenic  micro-organisms  may  be 
destroyed  by  the  gastric  juice.  The  common  bacillus  of  cholera,  certain 
streptococci,  etc.,  are  killed  by  the  gastric  juice,  while  others,  especiall}^ 
as  spores,  are  unacted  upon.  The  fact  that  gastric  juice  can  diminish  or 
retard  the  action  of  certain  toxalbumins,  such  as  tetanotoxine  and  diph- 
theria toxine,  is  also  of  great  interest  (Nexcki,  Sieber,  and  Schoumowa  ^). 

Because  of  this  antifermentative  and  antitoxic  action  of  gastric  juice  it 
is  considered  that  the  chief  importance  of  the  gastric  juice  lies  in  its  anti- 
septic action.  The  fact  that  intestinal  putrefaction  is  not  increased  on  the 
extirpation  of  the  stomach,  as  derived  from  experiments  made  on  man  and 
animals  2  does  not  uphold  this  view. 

*  Cohn,  Zeitschr.  f.  physiol.  Chem.,  14;  Strauss  and  Bialocour,  Zeitschr.  f.  klin. 
Med.,  2S.  See  also  Kiihne,  Lehrb.,  57;  Bunge,  Lehrb.  d.  Physiol.,  4.  Aufl.,  148  and 
1.59;  Hirschfeld,  Pfliiger's  Arch.,  47;  Nencki,  Sieber,  and  Schoumowa,  Centralbl.  f. 
Bacteriol..  etc.,  23.  In  regard  to  the  action  of  gastric  juice  upon  pathogenic  microbes 
we  must  refer  the  reader  to  handbooks  of  bacteriology. 

^  See  Carvallo  and  Pachon,  1.  c,  and  Schlatter  in  Wroblewski,  1.  c. 


372  DIGESTION. 

Since  the  hydrochloric  acid  of  the  gastric  juice  prevents  the  contents 
of  the  stomach  from  fermenting  with  the  generation  of  gas,  those  gases  which 
occur  in  the  stomach  probably  depend,  at  least  in  great  measure,  upon  the 
swallowed  air  and  saliva,  and  upon  those  gases  generated  in  the  intestine 
and  returned  through  the  pyloric  valve.  Planer  found  in  the  stomach- 
gases  of  a  dog  66-68  per  cent  N,  23-33  per  cent  CO2,  and  only  a  small 
quantity,  0.8-6.1  per  cent,  of  oxygen.  Schierbeck  ^  has  shown  that  a 
part  of  the  carbon  dioxide  is  formed  by  the  mucous  membrane  of  the 
stomach.  The  tension  of  the  carbon  dioxide  in  the  stomach  corresponds, 
according  to  him,  to  30-40  mm.  Hg  in  the  fasting  condition.  It  increases 
after  partaking  food,  independently  of  the  kind  of  food,  and  may  rise  to 
130-140  mm.  Hg  during  digestion.  The  curve  of  the  carbon-dioxide 
tension  in  the  stomach  is  the  same  as  the  curve  of  acidity  in  the  different 
phases  of  digestion,  and  Schierbeck  has  also  found  that  the  carbon-dioxide 
tension  is  considerably  increased  by  pilocarpine,  but  diminished  by  nico- 
tine. According  to  him,  the  carbon  dioxide  of  the  stomach  is  a  product 
of  the  activity  of  the  secretory  cells. 

i\fter  death,  if  the  stomach  still  contains  food,  autodigestion  goes  on 
not  only  in  the  stomach,  but  also  in  the  neighboring  organs,  during  the 
slow  cooling  of  the  body.  This  leads  to  the  question,  Why  does  the  stomach 
not  digest  itself  during  life?  Ever  since  Pavy  has  shown  that  after  tying 
the  smaller  blood-vessels  of  the  stomach  of  dogs  the  corresponding  part  of 
the  mucous  membrane  was  digested,  efforts  have  been  made  to  find  the 
cause  in  the  neutralization  of  the  acid  of  the  gastric  juice  by  the  alkali  of 
the  blood.  That  the  reason  for  the  non-digestion  during  life  is  to  be  sought 
for  in  the  normal  circulation  of  the  blood  cannot  be  contradicted;  but  the 
reason  is  not  to  be  found  in  the  neutralization  of  the  acid.  The  investi- 
gations of  Fermi  and  Otte  2  show  that  the  blood  circulation  acts  in  an 
indirect  manner  by  the  normal  nourishment  of  the  cell  protoplasm,  and  this 
is  the  reason  why  the  digestive  fluids,  the  gastric  juice  as  well  as  the  pan- 
creatic juice,  act  differently  upon  the  living  protoplasm  as  compared  with  the 
dead.  We  know  nothing  about  this  resistance  of  the  living  protoplasm. 
Some  claim  that  it  is  connected  closely  with  the  secretion  of  the  antipepsin 
discovered  by  Danilewsky,  Hansel,  and  Weinland,  but  this  is  hard  to 
understand.  Undoubtedly  bodies  occur  in  the  gastric  mucosa  which  can 
inhibit  the  action  of  pepsin,  but  whether  these  bodies  are  of  an  enzymotic 
nature  or  not  is  undecided.  Weinland's  antipepsin  is  related  to  the 
enzvmes  because  it  is  thermolabile,  while  the  antipepsin  of  Danilewsky, 


'  Planer,  Wien.  Sitzungsber.,  42;   Schierbeck,  Skand.  Arch.  f.  Physiol.,  3  and  5. 

^  Pa\y,  Phil.  Transactions,  153,  fart  I,  and  Guy's  Hospital  Reports,  13;  Otte, 
Travaux  du  laboratoire  de  I'lnstitut  de  Physiol,  de  Liege,  5,  1896,  which  also  con- 
tains the  literature. 


EXAAIINATIOX   OF   THE   GASTRIC    COXTENTS.  373 

Hansel,  and  0.  Schwahz  ^  is  resistant  towards  heat  and  can  hardly  be 
considered  as  an  enzymotic  body.  This  is  true  for  at  least  the  thermo- 
stabile  antipepsin  of  Schwaez,  which  does  not  give  the  biuret  reaction. 
Without  mentioning  the  still  unknown  nature  of  these  bodies,  the  natural 
gastric  juice,  as  well  as  an  acid  infusion  of  the  mucosa,  has  such  a  strong 
digestive  action  that  the  retarding  action  of  the  antipepsin  can  only  be 
shown  under  special  conditions,  and  it  is  therefore  difficult  to  conceive 
how  the  antipepsin  could  have  a  protective  action  in  life. 

Under  pathological  conditions,  irregularities  in  the  secretion  as  well  as  in 
the  absorption  and  in  the  mechanical  work  of  the  stomach  may  occur.  Pepsin 
is  almost  always  present,  although  the  amomit  may  vary  considerably,  but  the 
absence  of  the  rennin,  as  above  stated,  may  occur  in  many  cases.  In  regard 
to  the  acid,  we  must  remark  that  the  secretion  is  sometimes  uacreased  so  that 
an  abnormally  acid  gastric  juice  is  secreted  and  at  other  times  it  may  be  dimin- 
ished so  that  little  if  any  hydrochloric  acid  is  formed.  A  h}-persecretion  of 
acid  gastric  juice  sometimes  occurs.  In  the  secretion  of  too  little  hydrochloric 
acid  the  same  conditions  appear  as  after  the  neutralization  of  the  acid  contents 
of  the  stomach  outside  of  the  organism.  Fermentation  processes  now  appear  in 
which,  besides  lactic  acid,  there  occur  also  volatile  fatty  acids,  such  as  butjTic 
and  acetic  acids,  etc.,  and  gases  like  hydrogen.  These  fermentation  products  are 
therefore  often  found  in  the  stomach  in  cases  of  chronic  catarrh  of  the  stomach, 
which  may  give  rise  to  belching,  pjTosis,  and  other  s^Tnptoms. 

Among  the  foreign  substances  found  in  the  contents  of  the  stomach  we  have 
UREA,  or  ammonium  carbonate  derived  therefrom  in  uraemia;  blood,  which 
generally  forms  a  dark-brown  mass  tln'ough  the  presence  of  hsematiii,  due  to  the 
action  of  the  gastric  juice;  bile,  which,  especially  during  vomiting,  easily  finds 
its  way  through  the  pylorus  into  the  stomach,  but  whose  presence  seems  to  be 
without  hnportance. 

If  it  is  desired  to  test  the  gastric  juice  or  the  contents  of  the  stomach 
for  pepsin,  fibrin  may  be  employed.  If  this  is  thoroughly  washed  immedi- 
ately after  beating  the  blood,  well  pressed,  and  placed  in  glycerine,  it  may  be 
kept  in  serviceal^le  condition  for  an  indefinitely  long  time.  The  gastric 
juice  or  the  contents  of  the  stomach — the  latter,  if  necessaiy,  having  been 
previously  diluted  with  1  p.  m.  hydrochloric  acid — is  filtered  and  tested 
with  fibrin  at  ordinary'  temperature.  (It  must  not  be  forgotten  that  a 
control  test  must  be  made  with  acid  alone  and  another  portion  of  the  same 
fibrin.)  If  the  fibrin  is  not  noticeably  digested  within  one  or  two  hours, 
no  pepsin  is  present,  or  at  most  there  are  only  slight  traces. 

In  testing  for  rennin  the  liquid  must  be  first  carefully  neutralized.  To 
10  c.c.  of  unboiled,  amphoteric  (not  acid)  cow's  milk  add  1-2  c.c.  of  the 
filtered  neutralized  liquid.  In  the  presence  of  rennin  the  milk  should 
coagulate  to  a  solid  mass  at  the  temperature  of  the  body  in  the  course  of 
10-12  minutes  without  changing  its  reaction.  If  the  milk  is  diluted  too 
much  by  the  addition  of  the  liquid  of  the  stomach,  only  coarse  flakes  are 
obtained  and  no  solid  coagulum.  Addition  of  lime-salts  is  to  be  avoided, 
as  in  great  excess  the}'  may  produce  a  partial  coagulation  even  in  the 
absence  of  typical  rennin. 

In  many  cases  it  is  especially  important  to  determine  the  degree  of 

*  See  Hansel,  Biochem.  Centralbl.,  1.  p.  404,  and  2,  p.  326;  ^Yeinland,  Zeitschr.  f. 
Biologie,  44;   Schwarz,  Hofmeister's  Beitriige,  6. 


374  DIGESTION. 

acidity  of  the  gastric  juice.  This  may  be  done  ]:)y  the  ordinary  titration 
methods.  Phenolphthalein  must  not  be  used  as  an  indicator,  as  too  high 
results  are  produced  in  the  presence  of  large  quantities  of  proteins.  Good 
results  may  be  obtained,  on  the  contrary,  by  using  very  delicate  litmus  paper. 
Although  the  acid  reaction  of  the  contents  of  the  stomach  may  be  caused 
simultaneously  by  several  acids,  still  the  degree  of  acidity  is  here,  as  in 
other  cases,  expressed  in  only  one  acid,  e.g.,  HCl.  Generally  the  acidity 
is  designated  by  the  number  of  cubic  centimetres  of  N/10  caustic  soda 
which  is  required  to  neutralize  the  several  acids  in  100  c.c.  of  the  liquid 
of  the  stomach.  An  acidity  of  43  per  cent  means  that  100  c.c.  of  the  hquid 
of  the  stomach  required  43  c.c.  of  N/10  caustic  soda  to  neutrahze  it. 

It  is  also  important  to  be  able  to  ascertain  the  nature  of  the  acid  or 
acids  occurring  in  the  contents  of  the  stomach.  For  this  purpose,  and 
especially  for  the  detection  of  free  hydrochloric  acid,  a  great  number  of  color 
reactions  have  been  proposed  which  are  all  based  upon  the  fact  that  the 
coloring  substance  gives  a  characteristic  color  with  very  small  quantities 
of  hydrochloric  acid,  while  lactic  acid  and  the  other  organic  acids  do  not 
give  these  colorations,  or  only  in  a  certain  concentration,  which  can  hardly 
exist  in  the  contents  of  the  stomach.  These  reagents  are  a  mixture  of 
FERRIC-ACETATE  and  POTASsiuM-suLPHocYANiDE  solutiou  (Mohr's  reagent 
has  Ijeen  modified  by  several  investigators),  methylaniline-violet,  tro- 
p.EOLiN  00,  Congo  red,  malachite-green,  phloroglucin-vanillin, 
dimethylaminoazobenzene,  and  others.  As  reagents  for  free  lactic  acid 
Uffelmann  suggests  a  strongly  diluted,  amethyst-blue  solution  of  ferric 
CHLORIDE  and  carbolic  acid  or  a  strongly  diluted,  nearly  colorless  solu- 
tion of  ferric  chloride.  These  give  a  yellow  color  with  lactic  acid,  but 
not  with  hydrochloric  acid  or  with  volatile  fatty  acids. 

The  value  of  these  reagents  in  testing  for  free  hydrochloric  acid  or 
lactic  acid  is  still  disputed.  Among  the  reagents  for  free  hydrochloric  acid 
OiJNZBURG's  test  with  phloroglucin-vanillin,  and  the  test  with  tropseolin  00, 
performed  at  a  moderate  temperature  as  suggested  by  Boas,  and  the  test 
with  dimethylaminoazobenzene,  which  is  the  most  delicate,  seem  to  be  the 
most  valuable.  If  these  tests  give  positive  results,  then  the  presence  of 
hydrochloric  acid  may  be  considered  as  proved.  A  negative  result  does 
not  eliminate  the  presence  of  hydrochloric  acid,  as  the  delicacy  of  these 
reactions  has  a  limit,  and  also  the  simultaneous  presence  of  protein,  pep- 
tones, and  other  l^odies  influences  the  reactions  more  or  less.  The  reactions 
for  lactic  acid  may  also  give  negative  results  in  the  presence  of  compara- 
tively large  quantities  of  hydrochloric  acid  in  the  liquid  to  be  tested.  Sugar, 
sulphocyanides,  and  other  bodies  may  act  with  these  reagents  similarly  to 
lactic  acid. 

In  testing  for  lactic  acid  it  is  safest  to  shake  the  material  with  ether  and 
test  the  residue  after  the  evaporation  of  the  solvent.  On  the  evaporation 
of  the  ether  the  residue  may  be  tested  in  several  ways.  Boas  ^  utilizes  the 
property  possessed  by  lactic  acid  of  being  oxidized  into  aldehyde  and 
formic  acid  on  careful  oxidation  with  sulphuric  acid  and  manganese  dioxide. 
The  aldehyde  is  detected  by  its  forming  iodoform  with  an  alkaline  iodine 
solution  or  by  its  forming  aldehyde  mercury  with  Nessler's  reagent. 


*  Deutsch.  med.  Wochenschr.,  1893,  and  Miinchener  med.  Wochenschr,  1893. 


EXAMIXATIOX  OF  THE   G.\STRIC   COXTE^TS.  375 

The  quantitative  estimation  consists  in  the  formation  of  iodoform  with  X/10 
iodine  solution  and  caustic  potash,  adchng  an  excess  of  hydrochloric  acid  and 
titrating  ^\-ith  a  X/10  sodium-arsenite  solution,  and  retitrating  with  iodine  solu- 
tion, after  the  adrUtion  of  starch-paste,  until  a  blue  coloration  is  obtained.  This 
method  presupposes  the  use  of  ether  entirely  free  from  alcohol. 

In  testing  for  lactic  acid  Cro.veir  and  Croxheim  ^  recommend  a  solu- 
tion of  ioclij\e  in  potassium  iodide  containing  aniline.  Lactic  acid  is  con- 
verted mto  iodoform,  which  with  the  aniline  develops  the  nauseating  odor 
of  isonitrile.  The  shaking  out  of  the  lactic  acid  with  ether  is  imnecessarj", 
but  natm-ally  alcohol  or  acetone  must  not  be  added. 

In  order  to  be  able  to  judge  correctly  of  the  value  of.  the  different 
reagents  for  free  hydrochloric  acid,  it  is  naturally  of  greatest  importance  to 
be  clear  in  regard  to  what  we  mean  by  free  hychochloric  acid.  It  is  a 
weJJ-known  fact  that  hydrochloric  acid  combmes  wltn  proteins,  and  a 
considera]3le  part  of  the  h\'drochloric  acid  may  therefore  exist  m  the  con- 
tents of  the  stomach,  after  a  meal  rich  lq  proieixis.  m  combination  with 
them.  This  hydrochloric  acid  combined  with  proteins  cannot  be  con- 
sidered as  free,  and  it  is  for  this  reason  that  certain  investigators  consider 
such  methods  as  that  of  Sjoqvist,  wliich  will  be  described  below,  as  of  little 
value.  However,  it  must  be  remarked  that,  according  to  the  imanimous 
experience  of  many  investigators,  the  hydrochloric  acid  combmed  with 
proteins  is  physiologically  active.  Those  reactions  (color  reactions)  which 
respond  only  to  actual!^'  free  hydrochloric  acid  do  not  show  the  physiolog- 
ically active  tiydrochioric  acid.  The  suggestion  of  determining  the  "phys- 
iologically active"  hydrochloric  acid  instead  of  the  "'free"  seems  to  be 
correct  in  principle;  and  as  the  conceptions  of  free  and  of  physiologically 
active  hydrochloric  acid  are  not  the  same,  it  must  always  be  well  defiaed 
whether  one  wishes  to  determine  the  actually  free  or  the  physiologically 
active  hydrochloric  acid  before  any  conclusions  are  drawn  as  to  the  value 
of  a  certain  reaction. 

The  acid  reaction  may  be  partly  due  to  free  acid,  partly  to  acid  salts 
(monophosphates),  and  partly  to  both.  According  to  Leo^  one  can  test 
for  acid  phosphates  by  calcium  carbonate,  which  is  not  neutralized  there^ 
with,  while  the  free  acids  are.  If  the  gastric  content  has  a  neutral  reaction 
after  shaking  with  calcium  carbonate  and  the  carbon  dioxide  is  driven  out 
by  a  current  of  air.  then  it  contains  only  free  acid;  if  it  has  an  acid  reaction, 
then  acid  phosphates  are  present,  and  if  it  is  less  acid  than  before,  it  con- 
tains both  free  acid  and  acid  phosphate.  It  must  not  be  forgotten  that 
a  faint  acid  reaction  may.  after  treatment  with  calcium  carbonate,  also 
be  due  to  the  protein.  This  method  can  Iike"uise  be  applied  in  the  estima- 
tion of  free  acid. 

Various  titration  methods  have  been  suggested  for  the  estimation  of 
the  free  hydrochloric  acid,  but  these  cannot  yield  conclusive  results  for 
the  reasons  given  in  a  pre^■ious  chapter  (see  estimation  of  the  alkalinity 
of  the  blood-serum,  page  190).  For  this  determination  physico-chemical 
methods  are  necessar}-.  but  they  have  not  been  used  to  any  great  extent 
for  clinical  purposes  on  account  of  the  difficulty  in  their    manipulation. 

>  Berlin,  klin.  Wochenschr.,  190.5,  p.  lOSO. 

*CentraibL  f.  d.  med.  Wissensch.,lSS9,  p.  4S1;  Pfliiger's  Arch.,  -tS,  and  Berlin, 
klin.  "Wochenschr.,  1905,  p.  1491. 


376  DIGESTION. 

As  it  is  not  •vnthin  the  scope  of  this  l^ook  to  give  the  various  methods  for 
the  quantitative  estimation  of  h\-drochloric  acid  for  clinical  purposes  \vc 
must  refer  to  the  various  liandbooks  for  clinical  methods,  such  as  those 
of  V.  Jaksch,  Eulenburg,  Kolle,  and  Weixtraud,  and  the  work  of  O. 
Reissner,!  for  details  as  to  the  cjualitative  and  quantitative  tests  for 
hydrochloric  acid  and  lactic  acid. 

The  methods  suggested  by  Leo,  Hayem  and  Winter,  Martius  and 
LfrTTKE,  and  by  Reissxer,  as  well  as  the  following  method  of  Morxer 
and  Sjoqvist,2  are  used  for  tlie  quantitative  estimation  of  the  total  hydro- 
chloric acid. 

The  method  of  K.  IVroRXER  and  Sjoqvist  depends  upon  the  following  principle : 
When  the  gastric  juice  is  evaporated  to  chjniess  with  barium  carbonate  and  then 
calcined,  the  organic  acids  burn  up  and  give  insoluble  barium  carbonate,  while 
the  hychocliloric  acid  forms  soluble  barium  chloride.  From  the  quantity  of 
this  the  original  amount  of  hydrochloric  acid  can  be  calculated.  10  c.c.  of  the 
filtered  contents  of  the  stomach  are  mixed  in  a  small  platinum  or  silver  dish  with 
a  knife-point  of  bariiun  carbonate  free  from  clilorides  and  evaporated  to  dryness. 
The  residue  is  burned  and  allowed  to  glow  for  a  few  minutes.  The  cooled  carbon 
is  gently  rubbed  with  water  and  completely  extracted  with  boiling  water  and  the 
filtrate  (about  §0  c.c.)  precipitated  by  ammonium  chromate  after  the  addition  of 
ammonium  acetate  and  acetic  acid  and  boiling.  The  carefully  collected  precipi- 
tate is  washed  and  dissolved  in  water  by  the  aid  of  a  little  HCl,  treated  with  KI, 
and  hydrochloric  acid  and  titrated  with  hjqjosulphite  solution.  The  reactions  take 
place  as  follows-  4HC1 +2BaC03  =  2BaCl2  + 2H2O +2C0.;  2BaCl2+2(XHJoCr04  = 
2BaCrO,  +  4XH,C1 ;  2BaCrO,  +  16HC1  +  6KI  =  2BaCl2  +  Cvfk  +  8H2O  +  6KC1  +  3I2 ; 
and  3L_.  +  6Na2S203  =  6XaI  +  SXaaS^Ofi.  Each  cubic  centimetre  of  the  hyposul- 
phite corresponds  to  3  mgm.  HCl.  Complete  directions  for  the  necessary  solu- 
tions and  for  the  performance  of  the  method  may  be  found  in  SJOQ\^ST,  Zeitschr. 
f.  klin.  Med.,  32. 

In  testing  for  volatile  fatty  acids  the  contents  of  the  stomach  should  not 
be  directly  distilled,  as  volatile  fatty  acids  may  be  formed  by  the  decom- 
position of  other  bodies,  such  as  protein  and  haemoglobin.  The  neutral- 
ized contents  of  the  stomach  are  therefore  precipitated  with  alcohol  at 
ordinary  temperature,  filtered  quickly,  pressed,  and  repeatedly  extracted 
with  alcohol.  The  alcoholic  extracts  are  made  faintly  alkaline  by  soda 
and  the  alcohol  distilled.  The  residue  is  now  acidified  by  sulphuric  or 
phosphoric  acid  and  distilled.  The  distillate  is  neutralized  by  soda  and 
evaporated  to  dryness  on  the  water-bath.  The  residue  is  extracted  with 
absolute  alcohol,  filtered,  the  alcohol  distilled  off,  and  this  residue  dissolved 
in  a  }ittle  water.  This  solution  may  be  directly  tested  for  acetic  acid  with 
sulphuric  acid  and  alcohol  or  with  ferric  chloride.  Formic  acid  may  be 
tested  for  by  silver  nitrate,  which  quickly  gives  a  black  precipitate;  and 
butyric  acid  is  detected  by  the  odor  after  the  addition  of  an  acid.  In 
regard  to  the  methods  for  more  fully  investigating  the  different  volatile 
fatty  acids,  the  reader  is  referred  to  other  text-])Ooks. 

'Zeitschr.  f.  klin.  Med.,  48. 

^  In  regard  to  the  methods  here  mentioned  see  Reissner,  1.  c. 


INTESTINAL   GLANDS.  377 


III.  The  Glands  of  the  3Iucous  3Iembrane  of  the  Intestine  and  their 

Secretions. 

The  Secretion  of  Brunner's  Glands.  These  glands  are  partly  considered 
as  small  pancreatic  glands  and  partly  as  mucous  or  salivary  glands.  Their 
importance  in  various  animals  is  different.  According  to  Grutzner  they 
are  closely  related  in  dogs  to  the  pyloric  glands  and  contain  pepsin.  This 
also  coincides  with  the  observations  of  Glaessner  and  of  Poxoiviarew, 
which  differ  from  each  other  only  in  that  Ponomarew  finds  that  the  secre- 
tion is  inactive  in  alkaline  reaction  and  contains  only  pepsin,  while  Glaessxer 
claims  it  is  active  in  both  acid  and  alkaline  reaction  and  that  it  contains 
propepsin.  According  to  Abderhalden  and  Roxa  ^  the  pure  duodenal 
secretion  of  the  dog  contains  a  proteol}i:ic  enzyme  which  does  not  belong 
to  the  trypsm  type  but  rather  to  the  pepsin  variety.  The  statements  as 
to  the  occurrence  of  a  diastatic  enz^^me  in  Bruxxer's  glands  are  disputed. 

The  Secretion  of  Lieberkuhn's  Glands.  The  secretion  of  these  glands 
has  been  studied  by  the  aid  of  a  fistula  in  the  intestine  according  to  the 
method  of  Thiry  and  Vella.  Very  little  if  any  secretion  takes  place  in 
fasting  animals  (dogs)  when  the  mucous  membrane  is  not  irritated.  In 
Iambs  Pregl  found  the  secretion  continuous.  The  ingestion  of  food 
causes  a  secretion,  and  in  lambs  increases  the  secretion  already  taking 
place.  Mechanical,  chemical,  and  electrical  stimulants  act  in  the  same 
manner  in  dogs  (Thiry).  The  secretion  is  also  markedly  increased  in  man 
by  the  local  irritation  of  the  mucous  membrane  (Hamburger  and  Hekma^). 
In  the  cases  observed  bj'  these  experimenters  the  flow  of  fluid  was  greatest 
at  night  as  well  as  between  five  and  eight  o'clock  in  the  afternoon,  and 
was  lowest  between  two  and  five  o'clock  in  the  afternoon.  Pilocarpine 
does  not  increase  the  secretion  in  lambs,  and  in  dogs  it  does  not  seem  to 
be  always  active  (Gamgee^).  Among  the  chemical  excitants  we  must 
specially  mention  acids  and  gastric  juice,  which  latter  acts  by  its  acidity. 
The  action  of  acids  seems  to  be  indirect,  by  means  of  the  secretin  which 
will  be  mentioned  below.  Several  salts,  NaCl,  Na2S04,  and  others,  may 
cause  an  abimdant  secretion  of  fluid  into  the  intestine  when  injected  intra- 
venously or  subcutaneously,  as  well  as  after  direct  application  to  the  peri- 
toneal surface  of  the  intestine.     This  action  can  be  arrested  b}^  the  antag- 

*  Griitzner,  Pfliiger's  Arch.,  12;  Glaessner,  Hofmeister's  Beitrage,  1;  Ponomarew, 
Biochem.  Centralbl.,  1,  351;   Abderhalden  and  Rona,  Zeitschr.  f.  physiol.  Cliem.,  47. 

2  Thiry,  Wien.  Sitzungsber.,  50;  Vella,  Moleschott's  Untersuch.,  13;  Pregl,  Pfliiger's 
Arch.,  61;  Gamgee,  Physiol.  Chem.,  2,  410,  where  Vella  and  Masloff  are  quoted; 
Kruger,  Zeitschr.  f.  Biologie,  37;  Hamburger  and  Hekma,  Journ.  de  physiol.  et  de 
path,  generate,  1902  and  1904. 

^  Gamgee,  1.  c. 


378  DIGESTION. 

onistic,  inhibiting  action  of  a  lime  salt  (MacCallum  ^).  The  quantity  of 
this  secretion  in  the  course  of  twenty-four  hours  has  not  been  exactly 
determined. 

According  to  Delezenne  and  Frouin,  if  any  mechanical  irritation  is 
prevented,  the  fluid  flowing  spontaneously  from  a  fistula  in  a  dog  is  ten 
times  more  abimdant  in  the  duodenum  than  that  in  the  middle  or  lower 
part  of  the  jejunum.  In  the  upper  part  of  the  small  intestine  of  the  dog, 
on  the  contrary,  this  secretion  is  scanty,  slimy,  and  gelatinous;  in  the 
lower  part  it  is  more  fluid,  with  gelatinous  lumps  or  flakes  (Rohmann). 
Intestinal  juice  has  a  strong  alkaline  reaction  towards  litmus,  generates 
carbon  dioxide  on  the  addition  of  an  acid,  and  contains  (in  dogs)  nearly  a 
constant  quantity  of  NaCl  and  Na2C03,  4.8-5  and  4-5  p.  m.  respectively 
(GuMiLEWSKi,  Rohmann  2).  The  intestinal  juice  of  the  lamb  corresponded 
to  an  alkalinity  of  4.54  p.  m.  NaoCOa.  It  contains  protein  (Thiry  found 
8.01  p.  m.),  the  quantity  decreasing  with  the  duration  of  the  elimination. 
The  quantity  of  solids  varies.  In  dogs  the  quantity  of  solids  is  12.2-24.1 
p.  m.  and  in  lambs  29.85  p.  m.  The  specific  gravity  of  the  intestinal  juice 
of  the  dog,  according  to  the  observations  of  Thiry,  is  1.010-1.0107,  and 
in  lambs  1.01427  (Pregl).  The  intestinal  juice  from  lambs  contains 
18.097  p.  m.  protein,  1.274  p.  m.  proteoses  and  mucin,  2.29  p.  m.  urea, 
and  3.13  p.  m.  remaining  organic  bodies. 

We  have  the  investigations  of  Demant,  Turby  and  Manning,  H.  Ham- 
burger and  Hekma  and  Nagano  ^  on  the  human  intestinal  juice.  Human 
intestinal  juice  has  a  low  specific  gravity,  nearly  1.007,  about  10-14  p.  m. 
solids,  and  is  strongly  alkaline  towards  litmus.  The  content  of  alkali  calcu- 
lated as  sodium  carbonate  is  2.2  p.  m.,  according  to  Nagano,  Hamburger 
and  Hekma,  and  5.8-6.7  p.  m.  NaCl.  The  determination  of  the  freezing- 
point  was  —0.62°  (Hamburger  and  Hekma). 

The  intestinal  juice  of  the  dog  contains,  according  to  Boldireff,'*  a 
lipase  which  acts  especially  upon  emulsified  fat  (milk)  and  is  different  from 
pancreas  lipase.  The  intestinal  juice  of  animals  and  man  also  contains  an 
enzyme,  erepsin,  discovered  by  0.  Cohnheim,  which  does  not  act  ordinarily 
upon  native  proteins,  but  upon  proteoses  and  peptones,  and  the  juice  also 
has  a  faint  amylolytic  action.  The  juice,  and  to  a  high  degree  the  mucous 
coat,  contains  invertase  and  maltase,  which  fact  has  been  recently  substan- 
tiated by  the  observations  of  Paschutin,  Brown  and  Heron,  Bastianelli, 

»  University  of  California  Publications,  1,  1904. 

^  Delzenne  and  Frouin,  Compt.  rend.  soc.  biolog.,  56;  Gumilewski,  Pfliiger's  Arch., 
39;   Rohmann,  ibid.,  41. 

'Demant,  Virchow's  Arch.,  75;  Turby  and  Manning,  Centralbl.  f.  d.  med.  Wis- 
senschaft,  1892,  945;  Hamburger  and  Hekma,  1.  c;  Nagano,  Mitt,  aus  d.  Grenzgeb. 
d.  Med.  u.  Chir.,  9. 

*  Boldireff ,  Archiv  d.  sciences  biolog.  de  St.  Petersbourg,  11. 


INTESTINAL   JUICE.  379 

and  Tebb.^  A  lactose-inverting  enzyme,  a  lactase,  also  occurs,  as  shown 
by  RoHMANN  and  Lappe,  Pautz  and  Vogel,  Weinland,  and  Orban,2  in 
new-born  infants  and  young,  animals,  and  also  in  grown  mammals  which 
were  fed  upon  a  milk  diet.  The  lactase  is  found  to  a  greater  extent  in 
the  mucosa  than  in  the  juice. 

Besides  erepsin  and  the  other  enzymes  mentioned  the  intestinal  mucosa 
also  contains  antienzymes,  antipepsin  and  antitrypsin.  (Danilewsky  and 
Weinland  3),  also  enterokinase  or  a  mother-substance  of  the  same,  and 
finally  also  the  so-called  prosecretin.  These  two  last-mentioned  bodies, 
which  are  closely  connected  with  the  secretion  of  pancreatic  juice,  will 
be  discussed  in  connection  with  this  digestive  fluid. 

The  various  enzj-mes  are  not  formed  in  equal  quantities  in  all  parts  of 
the  intestine.  Lipase,  diastase,  and  invertase  occur,  according  to  Boldi- 
keff,  all  through  the  intestine,  while  the  kinase  occurs  only  in  the  upper  part 
of  the  intestine  (Boldireff,  Bayliss  and  Starling,  Delezenne).  Ac- 
cording to  Hekma  the  kinase  occurs  in  all  parts  of  the  intestine,  but  most 
abundantly  in  the  duodenum  and  the  upper  part  of  the  jejunum.  The 
enzymes,  according  to  Falloise,  generally  occur  in  greatest  abundance  in 
the  upper  parts  of  the  intestine;  but  the  erepsin  occurs  to  a  greater  extent  in 
the  jejunum  than  in  the  duodenum.  According  to  the  investigations  of  Ver- 
non the  behavior  of  erepsin  in  different  animals  is  not  the  same.  In  cats 
and  hedge-hogs  the  duodenum  is  richer  in  erepsin  than  the  jejunum  and 
ileum;  in  rabbits  it  is  the  reverse,  namely,  the  ileum  is  much  richer  than  the 
duodenum.  The  secretion,  according  to  Bayliss  and  Starling,  is  formed 
entirely  in  the  upper  part  of  the  intestine.  The  epithelium-cells  of  the  glands 
or  the  mucous  membrane  are  generally  considered  as  the  seat  of  forma- 
tion of  the  enzymes,  and  the  same  is  true  also  for  the  enterokinase,  according 
to  Bayliss  and  Starling,  Hekma,  Falloise,  and  others,  which,  however, 
according  to  Delezenne,*  is  formed  in  the  leucocj^es  and  Beyer's  glands. 

BoTTAZzi  5  has  obtained  a  very  complex  protein  from  the  intestinal  mucosa, 
which  is  readily  soluble  in  water  and  alkali  but  is  precipitated  by  acids.  It  coagu- 
lates at  55°  to  56°  and  probably  also  contains   carbohydrate    and  considerable 


'  Paschutin,  Centralbl.  f.  d.  med.  Wissensch.,  1870,  561;  Brown  and  Heron,  Annal. 
d.  Cliem.  u.  Pharm.,  204;  Bastianelli,  Moleschott's  Untersuch.  zur  Naturlehre,  11 
(ihis  contains  all  the  older  literature).  See  also  Miura,  Zeitschr.  f.  Biologie,  32;  Wid- 
dicombe,  Journ.  of  Pliysiol.,  28;  Tebb,  ibid.,  15. 

^  Rohmann  and  Lappe,  Ber.  d.  d-eutsch.  chem.  Gesellsch.,  28;  Pautz  and  Vogel, 
Zeitschr.  f.  Biologie,  32;  Weinland,  ibid.,  38;  Orban,  Maly's  Jahresber.,  29. 

^  See  foot-note  1,  p.  373. 

"Boldireff,  Arch.  d.  scienc.  biolog.  de  St.  Petersbourg,  11;  Bayliss  and  Starling, 
Journ.  of  Physiol.,  29,  30;  Hekma,  1.  c;  Falloise,  see  Biochem.  Centralbl.,  ■I,  p.  153; 
Vernon,  Journ.  of  Physiol.,  33;    Delezenne,  Compt.  rend.  soc.  biolog     51  and  56. 

'  See  Biochem.  Centralbl.,  3,  p.  65. 


380  DIGESTION. 

iron.  Intravenous  injection  of  this  protein  brings  about  an  abundant  secretion 
of  saliva,  pancreatic  juice,  bile,  and  intestinal  juice,  and  promotes  the  peristaltic 
movements  of  the  intestine. 

Erepsin.  This  enzyme,  discovered  by  O.  Cohnheim,  has  no  direct 
action  upon  native  proteins  with  the  exception  of  casein,  but  has  the  power 
of  splitting  proteoses  and  peptones.  In  this  change  mono-  as  well  as  di- 
amino-acids  are  produced.  Erepsin  occurs  in  the  mucous  membrane  and  in 
the  intestinal  juice  of  man  as  well  as  of  dogs;  the  mucous  membrane  seems 
to  be  richer  than  the  juice  (Salaskin,  Kutscher  and  Seemann  ^).  An 
enzyme  like  erepsin  occurs  also  in  the  pancreas  (Bayliss  and  Starling, 
Vernon),  and  this  has  the  power  of  acting  upon  casein,  but  not,  or  only 
faintly,  upon  fresh  fibrin.  This  erepsin  is  probably  identical  with  the 
enzyme  nuclease,  discovered  by  F.  Sachs  in  the  pancreas,  which  acts  upon 
nucleic  acids,  while  Nakayama  claims  that  erepsin  differs  from  trypsin 
by  having  a  cleavage  action  upon  nucleic  acids.  Erepsin  shows  a  great 
similarity  to  the  intracellular  enzymes  active  in  autolysis,  and  according  to 
Vernon  erepsins  occur  in  the  various  tissues  of  invertebrates  as  well  as 
vertebrates.  These  tissue  erepsins,  which  are  closely  related  to  the  auto- 
lytic  enzymes,  if  they  are  not  identical,  behave  somewhat  differently  from 
the  intestinal  erepsin  and  are  not  identical  therewith.  Enzymes  having 
an  action  similar  to  erepsin  occur,  according  to  Vines,^  in  all  plants  so  far 
investigated. 

Erepsin  becomes  inactive  on  heating  to  59°.  It  works  best  in  alkaline 
solution,  but  has  hardly  any  action  in  faint  acid  reaction.  In  this  regard, 
as  well  as  by  the  fact  that  only  a  little  ammonia  is  split  off  by  its  action 
upon  peptone  substances,  it  differentiates  itself  from  certain  of  the  auto- 
lytic  enzymes  studied  so  far. 

The  secretion  of  the  glands  in  the  large  intestine  seems  to  consist  chiefly 
of  mucus.  Fistulas  have  also  been  introduced  into  these  parts  of  the 
intestine,  which  are  chiefly,  if  not  entirely,  to  be  considered  as  absorption 
organs.  The  investigations  on  the  action  of  this  secretion  on  nutritive 
bodies  have  not  as  yet  yielded  any  positive  results. 

IV.    The  Pancreas  and  Pancreatic  Juice. 

In  invertebrates,  which  have  no  pepsin  digestion  and  which  also  have 
no  formation  of  bile,  the  pancreas,  or  at  least  an  analogous  organ,  seems  to 
be  the  essential  digestive  gland.  On  the  contrary,  an  anatomically  charac- 
teristic pancreas  is  absent  in  certain  vertebrates  and  in  certain  fishes. 

''Cohnheim,  Zeitschr.  f.  physiol.  Chem.,  33,  35,  36,  and  47;  Salaskin,  ibid.,  35; 
Kutscher  and  Seemann,  ibid.,  35. 

'  Bayliss  and  Starling,  Journ.  of  Physiol.,  30;  Vernon,  ibid.,  30  and  33;  F.  Sachs, 
Zeitschr.  f.  physiol.  Chem.,  46;  Nakayama,  ibid.,  41;  Vines,  Annals  of  Botany,  18  and 
19. 


PAIsCREAS.  381 

Those  functions  which  should  be  regulated  by  this  organ  seem  to  be  per- 
formed in  these  animals  by  the  Uver,  which  may  be  rightly  called  the  hepa- 
TOPAXCREAS.  In  man  and  in  most  vertebrates  the  formation  of  bile  and  of 
certain  secretions  containing  enzymes  important  for  digestion  is  divided 
between  the  two  organs,  the  liver  and  the  pancreas. 

The  pancreatic  gland  is  similar  in  certain  respects  to  the  parotid  gland. 
The  secreting  elements  of  the  former  consist  of  nucleated  cells  whose  basis 
forms  a  mass  rich  in  proteins,  which  expands  in  water  and  in  which  two 
distinct  zones  exist.  The  outer  zone  is  more  homogeneous,  the  inner  cloudy, 
due  to  a  quantity  of  granules.  The  nucleus  lies  about  midway  between  the 
two  zones,  but  this  position  may  change  with  the  var\'ing  relative  size  of 
the  two  zones.  According  to  Heidexil.\ix  ^  the  inner  part  of  the  cells 
diminishes  in  size  during  the  first  stages  of  digestion,  in  which  the  secretion 
is  active,  while  at  the  same  time  the  outer  zone  enlarges  owing  to  the 
absorption  of  new  material.  In  the  later  stage,  when  the  secretion  has 
decreased  and  the  absorption  of  the  nutritive  bodies  has  taken  place,  the 
inner  zone  enlarges  at  the  expense  of  the  outer,  the  substance  of  the  latter 
having  been  converted  into  that  of  the  former.  Under  physiological  con- 
ditions the  glandular  cells  are  undergoing  a  constant  change,  at  one  time 
consuming  from  the  inner  part  and  at  another  time  growing  from  the  outer 
part.  The  inner  granular  zone  is  converted  into  the  secretion,  and  the 
outer,  more  homogeneous  zone,  which  contains  the  repairing  material,  is 
then  converted  into  the  granular  substance.  The  so-called  islands  of 
Laxgerhaxs  are  related  to  the  internal  secretion  or  contain  a  substance 
taking  part  in  the  transformation  of  the  sugar  of  the  animal  body .2 

The  chief  portion  of  protein  substances  contained  in  the  gland  consists, 
it  seems,  of  micleoproteids,  while  the  globulins  and  albumins  occur  only  to  a 
slight  extent  as  compared  with  the  nucleoproteids.  Among  the  compound 
proteids  is  the  substance  studied  and  isolated  by  Uiiber  but  previously 
discovered  by  Hammarstex^  and  called  a-proteid.  This  nucleoproteid 
contains,  as  an  average.  1.67  per  cent  P.  1.29  per  cent  S,  17.12  per  cent 
N,  and  0.13  per  cent  Fe.  It  yields  on  boiling  _.3-proteid,  so  called  by  H.^i- 
M-\rstex,  which  is  much  richer  in  phosphorus  than  the  nucleoproteid. 
The  native  proteid  (a)  is  the  mother-substance  of  guanylic  acid;  accord- 
ing to  Umber  it  dissolves  by  pepsin  digestion  without  leaving  any  residue 
and  yields  on  tiypsin  digestion  guanylic  acid  on  one  side  and  proteoses 
and  peptones  on  the  other.     It  can  be  extracted  from  the  gland  by  a 


'  Pfluger's  .Ajch.,  10. 

^  See  Diamare  and  Kuliabko,  Centralbl.  f.  Physiol.,  18  and  19;  Rennie,  ibid.,  IS; 
Sauerbeck,  Virchow's  Arch...  177    Suppl. 

^  Umber,  Zeitschr.  f.  klin.  Med..,  40  and  43;  Hammarsten,  Zeitschr.  f.  physiol. 
Chem.,  19. 


382  DIGESTION. 

physiological  salt  solution  and  is  precipitated  by  acetic  acid.  Besides  this 
compound  proteid  the  pancreas  must  contain  at  least  one  other  proteid, 
which  is  the  mother-substance  of  the  thymonucleic  acid  obtainable  from 
the  pancreas. 

Besides  these  protein  substances  the  gland  contains  also  several  enzymes, 
or  more  correctly  zymogens,  which  will  be  discussed  later.  Among  the 
extractive  bodies,  which  are  probably  in  part  formed  by  post-mortem 
changes  and  chemical  action,  we  must  mention  leucine  (butalanine), 
tyrosine,  purine  bases  in  variable  quantities/  inosite,  lactic  acid,  volatile  fatty 
acids,  and  fats.  The  mineral  bodies  vary  considerably  in  quantity  not  only 
in  animals  and  man  but  also  in  men  and  women  (Gossmann),  The  calcium 
seems,  according  to  Gossmann,  to  exist  in  much  greater  amount  than  the 
magnesium.  According  to  the  investigations  of  Oidtmann  the  pancreas 
of  an  old  woman  contains  745.3  p.  m.  water,  245.7  p.  m.  organic  and  9.5 
p.  m.  inorganic  substances.  Gossmann  ^  found  in  a  man  17.92  p.  m.  ash 
and  13.05  p.  m.  in  a  woman. 

Besides  the  already-mentioned  (Chapter  VIII)  relationship  to  the  trans- 
formation of  sugar  in  the  animal  body,  the  pancreas  has  the  property  of 
secreting  a  juice  especially  important  in  digestion. 

Pancreatic  Juice.  This  secretion  may  be  obtained  by  adjusting  a  fistula 
in  the  excretory  duct,  according  to  the  methods  suggested  by  Bernard, 
LuDWiQ,  and  Heidenhain,  and  perfected  by  Pawlow.^  If  the  operation 
is  performed  with  sufficient  rapidity  and  under  favorable  conditions  a 
powerfully  active  secretion  may  be  obtained  either  immediately  after  the 
operation  (temporary  fistula)  or  after  some  time  (permanent  fistula). 

In  herbivora,  such  as  rabbits,  whose  digestion  is  uninterrupted,  the 
secretion  of  the  pancreatic  juice  is  continuous.  In  carnivora  it  seems,  on 
the  contrary,  to  be  intermittent  and  dependent  on  the  digestion.  During 
starvation  the  secretion  almost  stops,  but  commences  again  after  partaking 
of  food  and  reaches  its  maximum,  according  to  Bernstein, Heidenhain,  and 
others,  within  the  first  three  hours.  According  to  Pawlow  and  his  school 
(Walther  4)  this  maximum  is  dependent  upon  the  character  of  the  food. 
With  milk  diet  it  appears  within  three  to  four  hours,  after  bread  diet  at 
the  end  of  the  second  hour,  and  with  a  meat  diet  it  arrives  still  sooner. 
The  quality  of  the  juice  is  also,  according  to  Pawlow's  school,  dependent 

*  See  Kossel,  Zeitschr.  f.  physiol.  Chem.,  8. 

^  Gossmann,  Maly's  Jahresber.,  30;  Oidtmann,  cited  from  GoruD-Besanez,  Lehr- 
buch,  4thEd.,732. 

'  Bernard,  Legons  de  Physiol..  2,  190;  Ludwig,  see  Bernstein,  Arbeiten  a.  d.  physiol. 
.  Aristalt  zu  Leipzig,  1869;  Heidenhain,  Pfliiger's  Arch.,  10,  604;  Pawlow,  Die  Arbeit 
der  Verdauungsdrsiien,  Wiesbaden,  1898,  and  Ergebnisse  der  Physiologie,  1,  Abt.  1. 

*  Bernstein,  1.  c,  foot-note  3,  Walther,  Arch,  des  sciences  biol.  de  i  t. 
P^tersbourg,  7. 


PA^■CREATIC  JUICE.  383 

upon  the  food,  and  the  amount  of  the  three  enzymes,  diastase,  tn-psin,  and 
steapsin,  changes  with  the  variety  of  food.  The  observations  !R-hich  form, 
the  basis  of  this  view  have  been  somewhat  differently  explained  in  the  Ught 
of  recent  investigations  on  the  conditions  necessary  for  the  conversion  of 
trs-psinogen  into  tr\-psin. 

Pawlow   and  his   pupils,  especially   Schepowalxikoff.   have   shown, 
that  the  above-mentioned  (page  379)  enterokinase  activates  the  tr}psinogeii 
into  trvpsin.     These  observations  were  later  confirmed  by  others,  especially 
by  Delezen'XE  and  Frouix,  Popielski,  Camus  and  Gley.  Batliss  and 
Starling,  and  fiuther  studied.     The  pure  juice  contains  only  tr}-psinogen 
and  no  trypsin.     By  mixing  with  the  intestinal  juice,  or  by  contact  with  the 
intestinal  mucosa,  the  trypsinogen  is  converted  into  trypsin  by  the  kinase. 
Enterokinase,  which  itself  has  no  action  upon  proteins,  has  been  found  in 
all  higher  animals  examined.     A  kinase  with  a  similar  action  has  also  been 
detected  by  Delezexxe  in  the  lymph-glands  and  in  impure  fibrin,  a  state- 
ment which  is  contradicted  by  Batliss  and  Starling  and  Hekma.     The 
enterokinase  is  made  inactive  by  heat  and  is  therefore  considered  as  an 
enzyme.     Hamburger  and  Hekiia.  who  detected  enterokinase  in  human 
intestinal  juice,  do  not  con.-ider  it  an  enzyme,  because  a  certain  quantity 
of  intestinal  juice  will  activate  only  a  certain  quantity  of  trypsin  (see  below). 
The  above  statements  concerning  the  action  of  a  vars'ing  diet  upon  the 
enzyme  content  of  the  juice  have  been  somewhat  changed  by  the  investiga- 
tions of  Pawlow's  school  (Lintwarew  and  others).     For  instance,  a  diet  of 
bread  and  milk  causes  the  secretion  of  a  large  quantity  of  juice  which  is 
rich  in  trj'psinogen  but  contains  almost  no  trj'psin.     On  gi\"ing  meat  after 
this  the  juice  also  contains  tr\-psin;    after  a  rich  meat  diet  the  secretion 
becomes  scant  and  the  juice  contains  only  trs'psin  but  no  trypsinogen. 
There   is    here    one    difference    between    Pawlow's    school    and    certain 
other  investigators.     AccorcHng  to  Delezenne  and  Frouin,  Popielski, 
Bayliss   and   Starling,    Prym,    and    others,^   the   juice   never  contains 
trypsin  but  always  onlv  tr^-psinogen,  if  it  is  collected  through  a  canula  in 
Wirsung's  duct,  so  that  contact  with  the  intestinal  mucosa  is  prevented. 
Popielski  explains  the  ob.se rvat ions  of  Pa"«xow's  school  by  tlie  fact  that 
a  contact  of  the  juice  with  the  intestinal  secretions  was  not  perfectly  pre- 
vented, and  that  with  one  kind  of  diet  a  rapid  flow  of  jtiice  took  place  and 
with  another  a  slower  flow. 

It  is  not  clear  whether  there  are  also  kinases  for  the  other  two  enzymes. 
Pawlow's  pupils  claim  that  the  diastase  is  always  eliminated  as  enzyme, 
while  according  to  Pozerski  a  kinase  also  exists  for  this  zymogen.     In 

*  In  regard  to  the  literature  on  enterokinase,  secretin,  and  secretion  of  pancreatic 
juice,  see  O.  Cohnheim.  Biochem.  Centralbl.,  1,  169.  and  S.  Rosenberg,  ibid.,  2,  708; 
PrjTn,  Pfl   ger's  Arch.,  104  and  107. 


384  DIGESTION. 

regard  to  steapsin  the  statements  arc  somewhat  contradictory.  Accord- 
ing to  LiNTWAREW  there  is  secreted  with  food  rich  in  carbohydrates  and 
fats  a  zymogen  which  is  quickly  changed  into  the  enzyme  by  bile  or 
intestinal  juice.      With  a  meat  diet  the  steapsin  is  secreted  already  formed. 

The  specific  irritants  for  the  secretion  of  pancreatic  juice  are,  according 
to  Pawlow  and  his  collaborators,  acids  of  various  kinds — hydrochloric  acid 
as  well  as  lactic  acid — and  fats,  the  latter  acting  probably  by  virtue  of 
the  soaps  produced  therefrom.  Alkalies  and  alkali  carbonates  have,  on  the 
contrary,  a  retarding  action.  It  appears  that  the  acids  act  by  irritating 
the  mucosa  of  the  duodenum.  Water,  which  causes  a  secretion  of  acid 
gastric  juice,  likewise  becomes  indirectly  a  stimulant  for  the  pancreatic 
secretion,  but  may  also  be  an  independent  exciter.  The  psychical  moment 
may,  at  least  in  the  first  place,  have  an  indirect  action  (secretion  of  acid 
gastric  juice),  and  the  food  seems  otherwise  to  have  an  action  on  pancreatic 
secretion  by  its  action  on  the  secretion  of  gastric  juice. 

The  most  important  excitant  for  the  secretion  of  juice  is  hydrochloric 
acid,  but  the  views  are  not  united  as  to  the  manner  in  which  the  acid  acts 
According  to  Pawlow's  school,  the  acid  acts  reflexly  upon  the  intestine, 
causing  a  secretion  of  a  juice  containing  only  trypsinogen.  That  a  reflex 
action  is  here  produced  is  not  contradicted  by  the  investigations  of  Popiel- 
SKi,  Wertheimer  and  Lepage,  Fleig,i  and  others.  According  to  the 
researches  of  Bayliss  and  Starling,  which  have  been  confirmed  by  Camus, 
Gley,  Flei.g,  Herzex,  Delezenne,  and  others,  a  second  factor  must  also 
be  active  here.  Bayliss  and  Starling  have  shown  that  a  body  which 
they  have  called  secretin  can  be  extracted  from  the  intestinal  mucosa  by 
a  hydrochloric-acid  solution  of  4  p.  m. ,  and  this  when  introduced  into  the 
blood  produces  a  secretion  of  pancreatic  juice.  The  secretin,  which  accord- 
ing to  Bayliss  and  Starling  ^  is  the  same  in  all  vertebrates  examined,  is  not 
destroyed  by  heat;  it  is  therefore  not  identical  with  enterokinase,  and  is  not 
considered  as  an  enzyme.  It  is  formed  from  another  substance,  prosecretin, 
by  the  action  of  acids.  According  to  Delezenne  and  Pozerski^  secretin 
occurs  as  such  in  the  intestinal  mucosa,  and.  the  acid  acts  only  by  the 
destruction  of  certain  bodies  having  a  retarding  action.  According  to 
Popielski  secretin  action  is  different  from  acid  action;  the  secretin 
according  to  him  is  a  peptone,  and  the  secretin  action  can  also  be  obtained 
by  Witte's  peptone.  The  statements  about  secretin  and  its  action  are 
veiy   divergent.      It   is    diflficult    to    obtain   a   clear   conception   of  the 


^Gentralbl.  f.  Physiol.,  16,  681,  and  Compt.  rend.  soc.  biol.,  55.  See  also  foot- 
note l,p.  38.3. 

"^  Journ.  of  Physiol.,  29. 

^Delezenne  and  Pozei-ski,  Corapt.  rend.  soc.  biol.,  56;  Popielski,  Centralbl.  f. 
Physiol.,  19. 


PANCREAS  AND    ENZYME    FORMATION.  385 

amount  of  zymogens  or  enzymes  secreted  by  the  juice  under  the  in- 
fluence of  the  secretin.  It  seems  to  be  clear  that  this  juice,  at  least 
in  many  cases,  contains  only  trypsinogen  and  no  trj-psin. 

A  second  means  of  causing  secretion  is  the  fat,  which  probably  c^xy 
acts  after  it  has  been  saponified.  Oil-soap  introduced  directly  into  the 
duodenum  brings  about  a  strong  secretion  of  pancreatic  juice  (Sawitsch, 
Babkixe  ^),  and  at  the  same  time  a  flow  of  bile,  gastric  juice,  and 
the  secretion  of  Bruxxer's  glands  occurs.  The  pancreatic  juice  secreted 
imder  these  circumstances  has  about  the  same  amount  of  enzymes  as  the 
juice  secreted  after  partaking  of  food.  We  know  \ery  little  as  to  how  the 
soaps  act.  Fleig^  has  found  that  by  maceration  of  the  mucosa  of  the 
upper  part  of  the  duodenum  with  soap  solution  a  substance  goes  into 
solution,  which  he  calls  sapokrinin  and  which  when  mtroduced  into  the 
blood  brings  about  a  strong  secretion  of  pancreatic  juice.  This  sapo- 
krinin, which  is  derived  from  a  prosapokrinin,  is  not  an  enzyme  and  is  not 
identical  wdth  secretin.  It  dissolves  in  60  per  cent  alcohol  and  is  not 
destroyed  by  boiling.  Sapokrinin  affects  the  secretion  of  pancreatic  juice 
alone,  while  the  soaps  also  excite  the  secretion  of  bile  and  gastric  juice. 
The  secretion  of  pancreatic  juice  may  also  be  increased  by  alcohol  (Fleig, 

GiZELT  3). 

The  activation  of  the  tr}'psinogen  into  trypsin  may  in  life  be  brought 
about — as  the  researches  of  Herzex,  which  have  been  substantiated  b}^ 
Gachet  and  Pachon,  Bellamy,  jMendel  and  Rettger,  have  sho^Ti — not 
only  in  the  intestine,  but  also  in  the  gland  itself.  This  activation  of  the 
trj^psinogen  in  the  gland  itself  is  caused  in  a  manner  still  unknown  by  a 
body  of  unknowm  nature  formed  in  the  spleen,  which  is  congested 
during  digestion.  Such  a  "charging"  of  the  pancreas  by  the  spleen 
has  been  repeatedly  suggested  by  Schiff,^  but  this  has  recently  been  denied 
by  Prym.  According  to  this  experimenter  the  extirpation  of  the  spleen 
causes  no  change  in  the  properties  of  the  pancreatic  juice,  and  the  intra- 
venous injection  of  spleen  infusion  is  also  without  ac+ion  on  a  splenec- 
tomized  dog  with  permanent  pancreatic  fistula.  The  observations  of 
Herzen  that  a  spleen  infusion  has  a  strong  activating  action  upon  a 
weak  pancreas  infusion  were  sibstantiated  by  Pry:m,^  but  he  claims  that 
this  is  due  essentially  to  micro-organisms. 

The  conversion  of  the  tr}'psinogen  into  trj^psin  in  the  removed  gland  or 

^  Arch,  des  scienc.  biolog.  de  St.  Petersboiirg,  11. 
Compt.  rend.  soc.  biolog.,  55,  and  Journ.  de  physiol.  et  de  pathol.  g4n.,  1904. 

«  Centralbl.  f.  Physiol.,  19. 

*  Bellamy,  Joum.  of  Physiol.,  27;  Mendel  and  Rettger,  Amer.  Joum.  of  Physiol.,  7. 
A  very  complete  reference  to  the  literature  may  be  foimd  in  Menia  Besbokaia  Du 
rapport  fonctionell  entre  le  pankr6as  et  la  rate,  Lausanne,  1901. 

^  Pfliiger's  Arch.,  104  and  10.. 


386  DIGESTION. 

in  an  infusion  under  the  influence  of  air  and  water  and  also  by  other  bodies 
has  been  known  for  a  long  time.  According  to  ^'ERX0N  the  tr^'psin  itself 
has  a  strong  activating  action  upon  trypsinogen,  and  in  this  regard  it  is 
more  active  than  enterokinase.  The  correctness  of  this  statement  is  still 
denied  by  Bayliss  and  Starlixg  and  by  Hekma.  The  ordinary  view  of 
Heidexilun,  that  the  transformation  of  tr}-psinogen  into  trj'psin  is  also 
brought  about  by  acids,  has  been  found  to  be  incorrect  by  Hekma.^  Besides 
the  enterokinase  and  the  micro-organisms  we  know  for  the  present  of  no 
agent  of  organic  origin  which  has  the  power  of  activating  trypsinogen  with 
positiveness.  According  to  Delezenne,  on  the  contrary,  the  pancreatic 
juice  can  be  activated  by  calcium  salts,  and  according  to  E.  Zuxz  2  also  by 
magnesium  and  in  certain  cases  by  barium,  lithium,  and  strontium  salts. 

The  way  in  which  the  tr\^psinogen  is  converted  into  trypsin  is  still  un- 
knowTi  and  is  the  subject  of  disputed  \aews.  According  to  one  view,  pro- 
posed by  Pawlow  and  defended  by  Bayliss  and  Starlixg,  the  tryp- 
sinogen is  transformed  into  trj^psin  by  the  action  of  the  kinase.  According 
to  the  views  of  Delezenne,  Dastre  and  Stassaxo,  and  others,^  the  trypsin, 
on  the  contrar}^,  is  a  combination  between  the  kinase  and  trj-psinogen, 
analogous  to  the  hsemolysins,  which  according  to  Ehrlich's  side-chain 
theory  are  combinations  between  a  complement  and  an  amboceptor. 

The  reflex  formation  of  lactase  after  the  introduction  of  milk-sugar 
into  the  intestine,  as  observed  by  Weixlaxd,  is  to  be  considered  as  an 
intraglandular  enzyme  formation.  This  is  a  special  example  of  the 
general  rule  based  upon  Brocard's  researches,  that  the  kind  of  food  has 
a  marked  influence  upon  the  formation  of  hydrol}i;ic  ferments  in  the  body; 
"c'est  I'aliment  qui  fait  le  ferment."  It  has  not  been  determined  in  what 
way  the  milk-sugar  produces  this  adaptation  of  the  gland.  The  investiga- 
tions of  Baixbridge'*  seem  to  show  that  the  milk-sugar  causes  the  pro- 
duction of  a  body  in  the  intestinal  mucosa,  which  is  brought  to  the  pancreas 
by  the  blood  and  there  makes  the  formation  of  lactase  possible.  This 
special  property  of  the  pancreas  is  denied  by  Plimmer.^ 


'Vernon,  Journ.  of  Physiol.,  28;  Hekma,  Kon.  Akad.  v.  Wetenschappen  te 
Amsterdam,  190.3,  and  Arch.  f.  (Anat.  u.)  Physiol.,  1904;  BayUss  and  Starhng,  Journ. 
of  Physiol.,  30. 

^  Delezenne,  Compt.  rend.  ;-oc.  biolog.,  59,  and  Compt.  rend.,  141;  Zmiz,  see  Biochem. 
Centralbl.,  5,  69. 

'  Bayliss  and  Starling,  Journ.  of  Physiol.,  30  and  32,  which  also  cites  the  other 
investigators.     See  also  foot-note  1,  p.  383, 

*  Weinland,  Zeitschr.  f.  Biologie,  38  and  40;  Brocard,  Journ.  de  physiol.  et  de 
path,  gen.,  4;  Bainbridge,  Journ.  of  Physiol.,  31.  Contradictory  views  are  given 
by  Bierry,  Compt.  rend.,  140,  and  Compt.  rend.  soc.  biolog.,  58,  and  Pl.mmer,  Journ. 
of  Physiol.,  34. 

*  Journ.  of  Physiol.,  34. 


COMPOSITION  OF  PANCREATIC   JUICE.  387 

The  statements  as  to  the  quantity  of  pancreatic  juice  secreted  in  the 
twenty-four  hours  differ  very  much.  According  to  the  determinations 
of  Pawlow  and  his  collaborators,  Kuwschixski,  "Wassiliew,  and  Jablon- 
SKY,^  the  average  quantity  (with  normally  acting  juice)  from  a  permanent 
fistula  in  dogs  is  21.8  c.c.  per  kilo  in  the  twenty-four  hours. 

The  pancreatic  juice  of  the  dog  is  a  clear,  colorless,  and  odorless  alka- 
line fluid  which  when  obtained  from  a  temporary  fistula  is  ver}^  rich  in 
proteins,  sometimes  so  rich  that  it  coagulates  like  the  white  of  the  egg 
on  heating.  Besides  proteins  the  juice  contains  also  the  three  above- 
mentioned  enzymes  (or  their  zymogens),  amylopsin,  trypsin,  steapsin, 
also  an  enzyme  similar  to  erepsin,  and  besides  these  a  rennin,  which  was 
first  observed  by  Kuhxe.  Besides  the  above-mentioned  bodies  the  pan- 
creatic juice  habitually  contains  small  quantities  of  leucine,  fat,  and  soaps. 
As  mineral  constituents  it  contains  chiefly  alkali  chlorides  and  considerable 
alkali  carbonate,  some  phosphoric  acid,  lime,  magnesia,  and  iron. 

The  older  analyses  of  the  juice  from  a  permanent  fistula  by  C.  ScHAncT 
are  the  results  of  a  more  or  less  abnormal  secretion,  hence  we  shall  give  only 
the  analyses  of  juices  from  temporary  fistulas  on  dogs.^  The  results  are 
given  in  parts  per  1000. 

a.  b. 

Water 900.8  884.4 

Solids. 99.2  115.6 

Organic  substance 90.4  

Ash 8.8  

The  mineral  constituents  consisted  chiefly  of  XaCl,  7.4  p.  m.,  which  is  remark- 
able because  the  juice  contains  such  a  large  amount  of  alkali  carbonate.  In  the 
juice  examined  by  De  Zilwa  ^  the  quantity  of  alkali  in  the  secretin  juice  was 
5-7.9  p.  m.  and  in  the  pilocarpin  juice  2.9--5.3  p.  m.  Xa^COs. 

In  the  pancreatic  juice  of  rabbits  11-26  p.  m.  solids  have  been  found,  and  in 
that  from  sheep  14.3-36.9  p.  m.  In  the  pancreatic  juice  of  the  horse  9-15."  p.  m. 
solids  have  been  found ;  in  that  of  the  pigeon,  12- 14  p.  m. 

The  human  physiological  pancreatic  secretion  from  a  fistula  has  been 
investigated  by  Glaessxer.3  The  secretion  was  clear,  foamed  readil}-, 
had  a  strong  alkaline  reaction  even  towards  phenolphthalein,  and  contained 
globulin  and  albumin  but  no  proteoses  and  peptones.  The  specific  gravity 
was  1.0075  and  the  freezing-point  depression  was  J  = —0.46-0.49°.  The 
solids  were  12.44-12.71    p.  m..  the  total  protein   1.28-1.74  p.  m.,  and  the 

*  Arch,  des  sciences  de  St.  Petersbourg,  2,  .391.  The  older  statements  of  Keferstein 
and  Halhvachs.  Bidder  and  Schmidt,  and  others  may  be  found  in  K  ilme,  Lehrbuch, 
114. 

^  Cited  from  Maly  in  Hermann's  Handbucli  de  ■  Physiol.,  5.  Theil  II,  189. 

^  Journ.  of  Pliysiol.,  31. 

*Zeitschr.  f.  physiol.  Ciiem.,  40.  See  also  EUinger  and  Kohn,  ibid.,  45,  and  the 
investigations  upon  cystic  fluids  from  tlie  pancreas  by  Schumm,  ibid.,  36,  and  Murray 
and  Gies,  American  Medicine,  4,  1902. 


388  DIGESTION. 

mineral  bodies  5.66-6.98  p.  m.  The  secretion  contained  trypsinogen, 
which  was  activated  by  the  intestinal  juice.  Diastase  and  lipase  were 
present;  inverting  enzymes,  on  the  contrary,  were  not.  The  daily  quantity 
of  juice  was  500-800  c.c.  The  quantity  of  secretion,  of  ferments,  and  of 
alkalinity  was  lowest  in  starvation,  but  soon  rose  with  the  taking  of  food, 
and  reached  its  maximum  in  about  four  hours. 

Amylopsin  or  pancreatic  diastase,  which,  according  to  Korowin  and 
ZwEiFEL,  is  not  found  in  new-born  infants  and  does  not  appear  imtil  more 
than  one  month  after  birth,  seems,  although  not  identical  with  ptyalin,  to 
be  nearly  related  to  it.  Amylopsin  acts  very  energetically  upon  boiled 
starch,  and  according  to  Kijhne  also  upon  unboiled  starch,  especially  at 
37°  to  40°  C,  and  according  to  Vernon  ^  best  at  35°  C.  It  forms,  similar 
to  the  action  of  saliva,  besides  dextrin,  chiefly  isomaltose  and  maltose, 
with  only  very  little  dextrose  (Musculus  and  v.  Mering,  Kulz  and 
VoGEL^).  The  dextrose  is  probably  formed  by  the  action  of  the  invertin 
existing  in  the  gland  and  juice.  The  pancreatic  juice  of  the  dog  contains 
in  fact,  according  to  Bierry  and  Terroine,^  maltase,  whose  action  becomes 
apparent  only  after  very  faint  acidification  of  the  juice.  According  to 
Rachford  the  action  of  the  amylopsin  is  not  reduced  by  very  small  quan- 
tities of  hydrochloric  acid,  but  is  diminished  by  larger  amounts.  Vernon, 
GRtJTZNER,  and  Vv'achsmaxn  '^  find  that  the  action  is  indeed  accelerated  by 
very  small  quantities  of  hydrochloric  acid,  0.045  p.  m.,  while  alkalies  in 
very  small  amounts  have  a  retarding  action.  This  retarding  action  of 
alkalies  and  hydrochloric  acid  may  be  stopped  by  bile  (Rachford.) 

If  the  natural  pancreatic  juice  i?  not  to  be  obtained,  then  the  gland 
may  be  treated  with  water  or  glycerine.  This  infusion  or  the  glycerine 
extract  diluted  with  water  (when  glycerine  has  been  used  which  has  no 
reducing  action)  may  be  tested  directly  with  starch-paste.  It  is  safer, 
however,  to  first  precipitate  the  enzyme  from  the  glycerine  extract  by 
alcohol,  and  wash  with  this  liquid,  dry  the  precipitate  over  sulphuric  acid, 
and  extract  with  water.  The  enzyme  is  dissolved  by  the  water.  The 
test  for  sugar  may  be  performed  in  the  same  manner  as  in  the  saliva. 

Steapsin  or  Fat-splitting  Enzyme.  The  action  of  the  pancreatic  juice 
on  fats  is  twofold.  First,  the  neutral  fats  are  split  into  fatty  acids  and 
glycerine,  which  is  an  enzymotic  process;  and  secondly,  it  has  also  the 
property  of  emulsifying  the  fats. 

'Korowin,  Maly's  Jahresber.,  3;  Zweifel,  foot-note  1,  p.  344;  Kiihne,  Lehrbuch, 
117;   Vernon,  Journ.  of  Physiol.,  27. 

^  See  foot-note  4,  p.  344. 

'  See  Tebb,  Journ.  of  Physiol.,  15;  Bierry  and  Terroine,  Compt.  rend.  soc.  bio- 
log.  58. 

*  Rachford,  Amer.  Journ.  of  Physiol.,  2;  Vernon,  1.  c;  Griitzner,  Pfliiger's 
Arch.,  91. 


STEAPSIN.  389 

The  action  of  the  pancreatic  juice  in  splitting  the  fats  may  be  shown  in 
the  following  way:  Shake  olive-oil  with  caustic  soda  and  ether,  siphon  off 
the  ether  and  filter  if  necessary,  then  shake  the  ether  repeatedly  with  water 
and  evaporate  at  a  gentle  heat.  In  this  way  is  obtained  a  residue  of  fat 
free  from  fatty  acids,  which  is  neutral  and  which  dissolves  in  acid-free  alcohol 
and  is  not  coloreel  red  by  alkanet  tincture.  If  such  fat  is  mixed  with 
perfectly  fresh  alkaline  pancreatic  juice  or  with  a  freshly  prepared  infusion 
of  the  fresh  gland  and  treated  with  a  little  alkali  or  with  a  faintly  alkaline 
glycerine  extract  of  the  fresh  gland  (9  parts  glycerine  and  1  part  1  per  cent 
soda  solution  for  each  gram  of  the  gland),  and  some  litmus  tincture  added 
and  the  mixture  warmeel  to  37°  C,  the  alkaline  reaction  ■u  ill  gradually 
disappear  and  an  acid  one  take  its  place.  This  acid  reaction  depends  upon 
the  conversion  of  the  neutral  fats  by  the  enzyme  into  glycerine  and  free 
fatty  acids. 

The  splitting  of  the  neutral  fats  may  also  be  shown  more  exactly  by 
the  following  methoel:  The  mixture  of  neutral  fats  (absolutel}-  free  from 
fatty  acids)  and  pancreatic  juice  or  pancreas  infusion  is  digested  at  the  tem- 
perature of  the  body  and  treated  with  some  soda  and  repeatedly  shaken 
with  fresh  quantities  of  ether  until  all  the  unsplit  neutral  fats  are  removed. 
Then  it  is  made  acid  with  sulphuric  acid,  and  after  the  acid  liquor  has  been 
shaken  with  ether,  the  ether  is  evaporated,  and  the  residue  tested  for  fatty 
acids. 

Another  simple  process  for  the  demonstration  of  the  fat-splitting  action 
of  the  pancreatic  glands  is  the  following  (Cl.  Bernard)  :  A  small  portion  of 
the  perfectly  fresh,  fuiely  divided  gland  substance  is  first  soaked  in  alcohol 
(90  per  cent).  Then  the  alcohol  is  removed  as  far  as  possible  by  pressing 
between  blotting-paper,  after  which  the  pieces  of  gland  are  covered  with 
an  ethereal  solution  of  neutral  butter-fat  (which  may  be  obtained  by  shak- 
ing milk  with  caustic  soda  and  ether).  After  the  evaporation  of  the  ether 
the  pieces  of  gland  covered  with  butter-fat  are  pressed  between  two  w^atch- 
glasses  and  then  gently  heated  to  37°  to  4()°  C.  in  this  position.  After 
some  time  a  marked  oelor  of  butyric  acid  appears. 

The  action  of  the  pancreatic  juice  in  splitting  fats  is  a  process  analogous 
to  that  of  saponification,  the  neutral  fats  being  decomposed,  by  the  addition 
of  the  elements  of  water,  into  fatty  acids  anel  glycerine  according  to  the 
following  formula:  C3H5.O3.R3  (neutral  fat)  +3H20  =  C3H5.03.H3  (glycerme) 
+3(H.0.R)  (fatty  acid).  This  depends  upon  a  hydrolytic  splitting,  which 
was  first  positively  proved  by  Bernard  anel  Berthelot.  The  pancreas 
enzyme  also  decomposes  other  esters,  just  as  it  does  the  neutral  fats 
(Nexcki,  Baas).  The  fat -splitting  enzyme  of  the  pancreas  is,  according 
to  Pawt^ow  and  Bruxo,  aided  in  its  action  bj''  the  bile,  anel  according 
to  Engel  obeys  Schutz-Borissow's  rule  that  the  extent  of  cleavage 
during  a  given  time  is  proportional  to  the  square  root  of  the  quantity  of 
ferment.     The  investigations  of  Kaxitz  ^  have  led  to  the  same  results. 

'  Bernard,  Ann.  de  chini.  et  physique  (3),  25;  Berthelot,  Jahresber.  d.  Chem., 
1855,  733;  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  20;  Baas,  Zeitschr.  f.  physiol.  Chem., 
1-t,  416;  Bruno,  Arch,  des  sciences  biolog.  de  St.  Petersbourg,  7;  Engel,  Hofmeister's 
Beitrage,  7;  Kanitz,  Zeitschr.  f.  physiol.  Chem.,  46. 


390  DIGESTION. 

PoTTEViN  *  found  that  the  pancreas  (free  from  water)  coiild  form  olein  from 
oleic  acid  and  glycerine  It  is  claimed  that  the  gland  can  form  other  esters  from 
oleic  acid  or  stearic  acid  with  other  alcohols  (amyl  alcohol)  if  we  operate  only  in 
the  absence  of  water.  In  the  presence  of  considerable  water  the  pancreas  has  a 
reverse  saponifying  action. 

The  fatty  acids  which  are  sjDlit  off  by  the  action  of  the  pancreatic  jiiice 
combine  in  the  intestine  with  the  alkalies,  forming  soaps,  which  have  a 
strong  emulsifying  action  on  the  fats,  and  thus  the  pancreatic  juice  becomes 
cf  great  importance  in  the  emulsification  and  the  absorption  of  the  fats. 

Trypsin.  The  action  of  the  pancreatic  juice  in  digesting  proteins  was 
first  observed  by  Bernard,  but  first  proved  by  Corvisart.^  It  depends 
upon  a  special  enzyme  called  by  Kuhne  trj'psin.  This  enzyme,  as  previ- 
ously explained,  does  not  occur  in  the  gland  as  such  but  as  trj'psinogen. 
According  to  Albertoni  ^  this  zymogen  is  found  in  the  gland  in  the  last 
third  of  the  intra-uterine  life.  Enzymes  more  or  less  like  trypsin  occur 
in  other  organs  and  are  very  widely  diffused  in  the  vegetable  kingdom,* 
in  yeast  and  in  higher  plants,  and  are  also  formed  by  various  bacteria. 

As  we  know  of  so-called  antienzymes  for  other  enzymes,  so  we  also  have  anti- 
trypsins and  not  only  in  the  intestinal  canal  but  also  in  the  blood-serum.  The 
results  as  to  the  specificity  of  these  antitrypsins  in  various  animals,  as  well  as 
the  possibility  of  producing  antitrypsms  by  immunization  are  still  disputed 

Trypsin,  like  other  enzymes,  has  not  been  prepared  in  a  piu"e  condition. 
Nothing  is  positively  kno^ATi  in  regard  to  its  nature,  but  as  obtained  thus  far 
it  shows  a  variable  behavior  (KtJHXE,  Klug,  Levene,  Mays,  and  others). 
At  least  it  does  not  seem  to  be  a  nucleoproteid,  and  trj^psin  has  also  been 
obtained  which  did  not  give  the  biuret  test  (Klug,  Mays,  Schwarzschild). 
Trypsin  dissolves  in  water  and  glycerine,  while  Kuhxe's  trypsin  was  insol- 
uble in  glycerine.  It  is  very  sensitive  to  heat,  and  even  the  body  tempera- 
tiu'e  gradually  decomposes  it  (Vernon,  Mays).  In  neutral  solution  ifc 
becomes  inactive  at  45°  C.  In  dilute  soda  solution  of  3-5  p.  m.  it  is  still 
more  readily  destroyed  (Biernacki,  Vernon  ^).  The  presence  of  proteid 
or  proteoses  has,  to  a  certain  extent,  a  protective  action  on  heating  an 
alkaline  trj^psin  solution,  and  this  has  been  substantiated  by  recent  investi- 
gations of  Bayliss  and  Vernon.     The  simpler  cleavage  products  have  a  still 

1  Compt.  rend.,  138. 

^  Gaz.  hebdomadaire,  1857,  Nos.  15,  16,  19,  cited  from  Bunge,  Lehrbuch,  4.  Aufl., 
185. 

3  See  Maly's  Jahresber.,  8,  254. 

*  In  this  connection  see  Vines,  Annals  of  Botany,  16,  17,  18,  19,  and  Oppenheimer, 
Die  Fermente,  1900. 

'  Kiihne,  Verh.  d.  naturh.-med.  Vereins  zu  Heidelberg  (N.  F.),  1,  3;  E3ug,  Math 
naturw.  Ber.  avis  Ungam,  18,  1902;  Levene,  Amer.  Journ.  of  Physiol.,  5;  Mays, 
Zeitschr.  f.  physiol.  Chem.,  38;  Vernon,  Journ.  of  Physiol.,  28  and  29;  Biernacki. 
Zeitschr.  f.  Biologic,  28;   Schwarzschild,  Hofmeister's  Beitriige,  4,. 


TRYPSIN.  391 

greater  protective  action  (Vernon  ^).  Trypsinogen,  according  to  the 
unanimous  statements  of  several  experimenters,  is  more  resistant  towards 
alkalies  than  trypsin.  Trypsin  is  gradually  destroyed  by  gastric  juice 
and  even  by  digestive  hydrochloric  acid  alone. 

The  preparation  of  pure  trypsin  has  been  tried  by  various  experimenters. 
The  most  careful  work  in  this  direction  was  done  by  KtJHNE  and  Mays. 
Various  methods  have  been  suggested  by  Mays,  but  we  cannot  enter  into 
a  discussion  of  them.  A  very  pure  preparation  can  be  obtained  by  making 
use  of  the  combined  salting  out  with  NaCl  and  MgS04.  A  very  active 
solution,  and  one  that  can  be  kept  for  a  long  time  (for  more  than  twenty 
years  according  to  Hammarsten),  can  be  obtained  by  extracting  with 
glycerine  (HEmENHAiN  2).  An  impure  but  still  very  active  infusion  can 
be  obtained  after  a  few  days  b}^  allowing  the  finely  divided  gland  to  stand 
with  water  which  contains  5-lU  c.c.  chloroform  per  litre  (Salkowski)  at 
the  temperature  of  the  room.  This  infusion  can  be  kept  very  active  for 
several  years  at  the  cellar  temperature.  For  digestion  experiments  the 
active  commercial  trypsin  preparations  can  be  employed. 

Like  other  enzymes,  trypsin  is  characterized  by  its  action,  and  this 
action  consists  in  dissolving  protein  and  in  splitting  it  into  simpler  products, 
mono-  and  diamino-acids,  trj^ptophane,  etc.,  in  alkaline,  neutral,  and  indeed 
in  very  faintly  acid  solutions.  This  action  has  been  so  far  considered  as 
characteristic  for  trypsin.  Recent  investigations  seem  to  indicate  that 
this  action  is  not  due  to  one  enzyme  alone  but  to  the  combined  action 
of  several  enzymes. 

There  is  no  question  that  in  the  pancreas  there  occurs  besides  trypsin 
also  an  enzyme  similar  to  erepsin  (Bayliss  and  Starling,  Vernon  3). 
According  to  the  latter  this  erepsin  has  a  strong  action  upon  peptone, 
and  he  believes  that  the  peptone-splitting  action  of  a  pancreas  infusion  is 
in  great  part  due  to  the  erepsin.  The  pancreas  besides  these  also  contains 
a  nuclease  (see  page  380),  whose  relationship  to  pancreas  erepsin  has  not 
been  determined. 

The  unity  of  trj'psin  has  also  been  disputed  from  another  point  of  view. 
According  to  Pollak  the  trypsin  (in  the  ordinary  sense)  contains  a  second 
enzyme,  which  does  not  act  upon  protein  but  only  upon  gelatine,  and  he 
calls  this  enzyme  glutitiase.  This  glutinase  is  much  more  resistant  towards 
acids  than  trypsin,  and  by  proper  treatment  with  acids  Pollak ^  was  able 
to  change  a  pancreas  infusion  so  that  it  acted  upon  gelatine  and  not  upon 
certain  proteins.     The  correctness  of  these  statements  has,  indeed,  not 

*  Bayliss,  Arch,  des  scienc.  biolog.  de  St.  Petersbourg,  11,  Suppl.;  Vemon,  Joum. 
of  Physiol.,  31. 

'  Pfliiger's  Arch.,  10. 

'  Bayliss  and  Starling,  Joum.  of  Physiol.,  30;  Vernon,  ibid.,  30. 

*  Hofmeister's  Beitrage,  6.  Contradictory  statements  may  be  found  in  Ehren- 
reich,  cited  in  Biochem.  Centralbl.,  4. 


392  DIGESTION. 

been  generally  accepted;  nevertheless,  we  have  here  a  warning  to  be  care- 
ful as  to  the  conclusions  drawn  from  results  where  impure  infusions  are 
used.  For  many  experiments  it  is  undoubtedly  advisable  to  use  the  natural 
pancreatic  juice. 

As  in  recent  times  the  unity  of  trj'psin  has  been  in  doubt,  the  foUovring 
statements  apply  only  to  the  enzyme  which  we  have  been  in  the  habit  of 
calling  trypsin. 

The  action  of  trypsin  on  proteins  is  best  demonstrated  by  the  use  of 
fibrin.  Very  considerable  quantities  of  this  protein  body  are  dissolved 
by  a  small  amoimt  of  trypsin  at  37-40°  C.  It  is  always  necessary  to  make 
a  control  test  with  fibrin  alone,  with  or  without  the  addition  of  alkali. 
Fibrin  is  dissolved  by  trypsin  without  any  putrefaction;  the  liquid  has  a 
pleasant  odor  somewhat  like  bouillon.  To  completely  exclude  putrefac- 
tion a  little  thymol,  chloroform,  or  toluene  should  be  added  to  the  liquid. 
Tryptic  digestion  differs  essentially  from  pepsin  digestion,  irrespective  of 
the  difference  in  the  digestive  products,  in  that  the  first  takes  place 
in  neutral  or  alkaline  reaction  and  not,  as  is  necessary  for  peptic  digestion, 
in  an  acidity  of  1-2  p.  m.  HCl,  and  further  by  the  fact  that  the  proteins 
dissolve  in  trypsin  digestion  without  previously  swelling  up. 

As  trypsin  not  only  dissolves  proteids,  but  also  other  protein  substances 
such  as  gelatine,  this  latter  body  may  be  used  in  detecting  trj^psin.  The 
liquefaction  of  strongly  disinfected  gelatine  is,  according  to  Fermi, ^  a 
very  delicate  test  for  trypsin  or  tryptic  enzymes.  Various  suggestions  for 
the  use  of  gelatine  in  the  trypsin  test  have  been  made,  but  in  considera- 
tion of  the  above  statements  of  Pollak  in  regard  to  glutinase  it  is  prob- 
ably best  for  the  present  to  discard  the  use  of  gelatine  in  detecting  trj^psin. 

For  the  quantitative  estimation  of  trypsin  by  measuring  the  rapidity  of 
digestion  we  generally  make  use  of  the  method  of  Mett,  described  under  pepsin, 
digestion.  Another  method,  suggested  by  Weiss,  consists  in  determining  the 
nitrogen  in  the  filtrate  after  coagulation  with  heat  and  acetic  acid.  Lohlein 
recommends  the  titration  method  of  Volhard  as  used  in  pepsin  determinations, 
and  has  given  directions  for  its  use.^ 

Many  circumstances  exert  a  marked  influence  on  the  rapidity  of  the 
trypsin  digestion.  With  an  increase  in  the  quantity  of  enzyme  present  the 
digestion  is  hastened,  at  least  to  a  certain  point.  According  to  Pawlow  and 
his  school,  the  rule  of  ScHiJTZ-BoRissow  is  perfectly  applicable  to  tr^'psin, 
and  the  amount  digested  is  proportional  to  the  square  root  of  the  quantity  of 
ferment.  Based  upon  the  investigations  of  Bayliss,  Hedin,  and  Lohlein,^ 
this  assumption  does  not  seem  to  have  sufficient  foundation,  and  further 

'  Arch.  f.  Hygiene,  12  and  55. 

2  Weiss,  Zeitschr.  f.  physiol.  Chem.,  40;  Lohlein,  Hofmeister's  Beitrage,  7. 

^  Pawlow,  Die  Arbeit  der  Verdauungsdriisen,  Wiesbaden,  189S,  p.  33;  Bayliss, 
Arch,  des  scienc.  biolog.  de  St.  P^tersbourg,  11,  Suppl.;  Hedin,  Journ.  of  Physiol., 
32;  Lohlein,  1.  c. 


ACTION  OF  TRYPSIN.  393 

exp:  riments  in  this  direction  are  verj-  desirable,  as  so  far  experimenters  have 
worked  with  pancreas  infusions  or  commercial  trypsin  preparations,  which 
are  generally  impure  mixtures  of  enzj-mes.  Tryptic  digestion  is  also  acceler- 
ated by  an  increase  of  temperature,  at  least  to  about  40°  C,  at  which  tem- 
perature the  protein  is  very  rapidly  dissolved  by  the  trypsin.  The  reaction 
is  also  of  the  greatest  importance.  Trypsin  acts  energetically  in  neutral, 
cr  still  better  in  alkaline,  solutions,  and  best  in  an  alkalinity  of  3-4  p.  m. 
Na2C03;  but  the  nature  of  the  protein  is  also  of  importance.  The  action 
of  the  alkali  depends  upon  the  number  of  hydroxyl  ions  (Dietze,  Kaxitz), 
and  according  to  Kaxitz^  the  digestion  proceeds  best  in  those  solutions 
which  are  1/70-1/200  normal  in  regard  to  hydroxyl  ions.  Free  mineral 
acids,  even  in  verv'  small  quantities,  completely  prevent  the  digestion.  If  the 
acid  is  not  actually  free,  but  combined  with  protein  bodies,  then  the  diges- 
tion may  take  place  quickly  when  the  acid  combination  is  not  in  too  great 
excess  (Chittexdex  and  Cummixs).  Organic  acids  act  less  disturb ingl}-, 
and  in  the  presence  of  0.2  p.  m.  lactic  acid  and  the  simultaneous  pres- 
ence of  bile  and  common  salt  the  digestion  may  indeed  proceed  more  quickly 
than  in  a  faintly  alkaline  liquid  (Lixdberger).  The  statement  of  Rach- 
FORD  and  Southgate,  that  the  bile  can  prevent  the  injurious  action  of 
the  hydrochloric  acid,  and  that  a  mixture  of  pancreatic  juice,  bile,  and 
hydrochloric  acid  digests  better  than  a  neutral  pancreatic  juice,  could 
not  be  substantiated  by  Chittexdex  and  Albro.  That  bile  has  an  action 
tending  to  aid  the  trs-ptic  digestion  has  been  shown  by  many  investigators 
and  recently  by  Bruxo,  Zuxtz  and  Ussow.^ 

Carbon  dioxide,  according  to  Schierbeck,^  has  a  retarding  action  in 
acid  solutions,  but  it  accelerates  the  tryptic  digestion  in  faintly  alkaline 
liquids.  Foreign  bodies,  such  as  borax  and  potassium  cyanide,  maj'  pro- 
mote trj'ptic  digestion,  while  other  bodies,  such  as  salts  of  mercmy,  iron, 
and  others  (Chittexdex  and  Cummixs),  or  salicylic  acid  in  large  quan- 
tities, may  have  a  preventive  action.  According  to  Weiss  "*  the  halogen 
alkali  salts  disturb  trv'ptic  digestion  only  slightl}^  and  NaCl  seems  to  have 
the  strongest  action.  The  sulphates  have  a  much  stronger  retarding  action 
than  the  chlorides.  Borax  had  no  influence,  while  sodium  phosphate,  on 
the  contrary,  had  a  strong  accelerating  action.  The  nature  of  the  -proteins 
is  also  of  importance.     Unboiled  fibrin  is,  relatively  to  most  other  proteins, 

*  Kanitz,  Zeitschr.  f.  physiol.  Chem. ,  3",  who  also  cites  Dietze. 

^  Chittenden  and  Cummins,  Studies  from  the  Physiol.  Chem.  Laboratory  of  Yale 
College,  New  Haven,  1SS5,  1,  100;  Lindberger,  Maly's  Jahresber.,  13;  Rachford  and 
Southgate,  Medical  Record,  1S95;  Chittenden  and  Albro,  Amer.  Joum.  of  Physiol.,  I, 
1898;  Rachford,  Joum.  of  Physiol.,  25;  Bruno,  1.  c;  Zvmtz  and  Ussow,  Arch.  f.  (Anat. 
u.)  Physiol.,  1900. 

^  Skand.  Arch.  f.  Physiol.,  3. 

M.  c. 


394  DIGESTION. 

dissolved  so  very  quickly  that  the  digestion  test  with  raw  fibrin  gives  an 
incorrect  idea  of  the  power  of  trypsin  to  dissolve  coagulated  protein  bodies 
in  general.  Boiled  fibrin  is  digested  with  much  greater  difficulty  and 
requires  also  a  higher  alkalinity:  8  p.  m.  NagCOs  (Vernon  ^).  The  resist- 
ance of  certain  native  protein  solutions,  such  as  blood-serum  and  egg-white, 
against  the  action  of  trypsin  is  remarkable;  a  behavior  which  can  be 
explained  by  the  occurrence  of  antitrypsin  in  these  solutions.  An  accumu- 
lation of  the  products  of  digestion  tends  to  hinder  the  trypsin  digestion. 

The  Products  of  the  Tryptic  Digestion.  In  the  digestion  of  unboiled 
fibrin  a  globulin  which  coagulates  at  55-60°  C.  may  be  obtained  as  an 
intermediate  product  (Herrmann  2),  Besides  this  one  obtains  from  fibrin, 
as  well  as  from  other  proteins,  the  products  previously  mentioned  in  Chapter 
II.  In  trypsin  digestion  the  cleavage  may  proceed  so  far  that  the  mix- 
ture fails  to  give  the  biuret  reaction.  This  does  not  indicate,  as  E.  Fischer 
and  Abderhalden  have  shown,  a  complete  cleavage  of  the.  protein  mole- 
cule into  mono-  and  diamino-acids,  etc.  In  tryptic  digestion,  as  shown  by 
Abderhalden  and  Reinbold,3  using  the  protein  edestin,  a  gradual  cleavage 
of  the  protein  takes  place,  and  thereby  certain  amino-acids,  like  tyrosine  and 
tryptophane,  are  readily  and  completely  split  off,  while  others,  like  leucine, 
alanine,  aspartic,  acid,  and  glutamic  acid,  are  slowly  and  less  readily  split 
off,  and  others,  such  as  a-proline,  phenylalanine,  and  glycocoU,  stubbornly 
resist  the  cleavage  action  of  the  trypsin.  The  polypeptide-like  bodies  dis- 
covered by  Fischer  and  Abderhalden,  which  are  produced  in  digestion, 
and  which  do  not  give  the  biuret  reaction,  are  the  atomic  complexes  which 
resist  the  action  of  trypsin.  These  polypeptides  contain  the  pyrrolidine 
carboxylic  acid  and  phenylalanine  groups  of  the  protein,  but  also  yield 
other  monamino-acids  such  as  leucine,  alanine,  glutamic  acid,  and  aspartic 
acid.  In  tryptic  digestion  no  more  nitrogen  is  split  off  as  ammonia  than 
on  hydrolysis  with  acids  (Mochizuki),  which  is  a  difference  between  trypsin 
and  the  autolytic  enzymes.  Among  the  above-mentioned  products  we  find 
on  the  autodigestion  of  the  gland  other  substances,  such  as  oxyphenyl- 
ethylamine  (Emerson),  which  is  produced  from  tyrosine  by  fermentive  CO2 
cleavage;  also  uracil  (Levene),  guanidine  (Kutscher  and  Otori),  the 
purine  bases,  which  originate  from  the  nuclein  bodies,  and  choline,  which 
latter  is  formed  from  lecithin  (Kutscher  and  Lohmann  4).  If  putrefaction 
is  not  completely  prevented,  still  other  bodies  occur  which  will  be  con- 
sidered later  in  connection  with  the  putrefactive  processes  in  the  intestine. 

»  Journ.  of  Physiol.,  28. 

^  Herrmann,  Zeitschr.  f.  physiol.  Chem.,  11. 

2  Zeitschr.  f.  physiol.  Chem.,  11  and  16.     See  also  Chapter  II. 

•*  Fischer  and  Abderhalden,  Zeitschr.  f.  physiol.  Chem.,  31);  Mochizuki,  Hofmeister's 
Beitrage,  1;  Emerson,  ibid.,  1;  Levene,  Zeitschr.  f.  physiol.  Chem.,  37;  Kutscher  and 
Lohmann,  ibid.,  39;   Kutscher  and  Otori,  ibid.,  13,  and  Centralbl.  f.  Physiol.,  18. 


ACTION    OF   TRYPSIN.  395 

The  Action  of  Trypsin  upon  other  Bodies.  The  nucleoproteids  and  nucleins 
are  so  digested  that  the  proteid  complex  is  separated  from  the  nucleic 
acid  and  then  digested.  The  nucleic  acids  may,  nevertheless,  be  somewhat 
changed  (Araki),  which  is  probably  brought  about  by  another  enzyme, 
the  nuclease  (Sachs).  A  cleavage  of  nucleic  acids  with  the  setting  free  of 
phosphoric  acid  and  purine  bases  is,  according  to  Iwanoff,^  not  brought 
about  by  trypsin.  This  splitting  is  first  produced  by  the  action  of  nuclease 
or  erepsin  (see  page  380).  Gelatine  is  dissolved  and  digested  by  pancreatic 
juice.  A  cleavage  with  the  separation  of  glycocoU  and  leucine  does  not 
occur  (KtJHNEand  Ewald),  or  only  to  a  trivial  extent  (Reich-Herzberge^). 

The  gelatine-forming  substance  of  the  connective  tissues  is  not  directly 
dissolved  by  trypsin,  but  only  after  it  has  been  treated  with  acids  or  soaked 
in  water  at  70°  C.  By  the  action  of  trypsin  on  hyaline  cartilage  the  cells 
dissolve,  leaving  the  nucleus.  The  matrix  is  softened  and  shows  an  indis- 
tinctly constructed  network  of  collagenous  substance  (KDhne  and  Ewald). 
The  elastic  substance,  the  structureless  membranes,  and  the  membrane  of  the 
fat-cells,  are  also  dissolved.  Parenchymatous  organs,  such  as  the  liver  and 
the  muscles,  arc  dissolved  all  but  the  nuclei,  connective  tissue,  fat-cor- 
puscles, and  the  remainder  of  the  nervous  tissue.  If  the  muscles  are  boiled, 
then  the  connective  tissue  is  also  dissolved.  Mucin  is  dissolved  and  split 
by  trypsin,  while  chili n  and  horn  substance  do  not  seem  to  be  acted  upon 
by  the  enzyme.  Oxyhmnoglobin  is  decomposed  by  trypsin  with  the  split-- 
ting  off  of  haematin.     Trj-psin  has  no  action  upon  fats  and  carbohydrates. 

We  have  the  investigations  of  Gulewitsch,  Gonneriviaxn,  Schwarz- 
scHiLD,3  E.  Fischer  and  Bergell,  and  Abderhalden  ^  upon  the  action  of 
trypsin  on  simply  constructed  substances  of  knoiMi  constitution,  such  as 
acid  amides  and  several  others  that  give  the  biuret  reaction.  An  undoubted 
cleavage  on  Curtius's  biuret  base  was  first  observed  by  Schwarzschild. 
The  investigations  of  Fischer  and  his  co-workers  are  much  more  complete 
and  important.  From  these  it  is  shown  that  the  pancreatic  juice  splits  a 
large  number  of  peptides,  as  well  as  di-  and  tri-  or  tetrapeptides,  while 
it  is  without  action  upon  a  large  number  of  others.  The  structure 
of  these  plays  an  important  role,  as,  for  example,  alanyl-glycine, 
CH3.CH(NH2).C0.NH.CH2.C00H,  is  split,  while  its  isomereglycyl-alanine, 
NH2.CH2.CO.NH.CH(CH3).COOH,  is,  on  the  contrarj^  not  split.  The 
nature  of   the  amino-acids  existing  in  the  peptide  is  also  of  importance. 

•  Iwanoflt,  Zeitschr.  f.  physiol.  Chem.,  39,  which  also  contains  the  literature;  Sachs, 
Hid.,  46. 

^  Kiihne  and  Ewald,  Verh.  d.  naturh.-med.  Vereins  zu  Heidelberg  (N.  F.),  1;  Reich- 
Herzberge,  Zeitschr.  f.  physiol.  Chem.,  84. 

'  Hofmeister's  Beitrage,  4,  where  the  other  works  are  also  cited. 

*  Fischer  and  Bergell,  Ber.  d.  d.  chern.  Gesellsch.,  Z?t  and  37;  Fis^^her  and  Abder- 
halden, Sitzungsber.  der  Kgl.  Pr.  Akad.  d.  Wissensch.,  Berlin,  1905. 


396  DIGESTION. 

Those  dipeptides  which  contain  alanine  as  acyl — for  example,  alanyl- 
glycine,  alanyl-alanine,  and  alanyl-leucine  A — are  readily  hydrolyzed,  while 
several  dipeptides  in  which  a-aminoljutyric  acid  or  leucine  functionates  as 
acyl  are  \ery  resistant.  The  number  of  amino-acid  groups  is  also  of 
importance,  as,  for  example,  triglj'cyl-glycine  is  not  split,  while  tetraglycyl- 
glycine  is.  In  those  peptides  which  are  racemic  bocUes  the  hydrolysis  takes 
place  asymmetrically,  so  that  only  one  half  of  the  racemic  body  is  attacked, 
and  those  active  amino-acids  result  as  products  whicli  are  contained  in  the 
natural  protein  bodies.  Tiiis  hydrolysis  of  various  pol3-peptides  by  means 
of  pancreatic  juice  is  of  especially  great  interest  from  several  points  of 
\'iew. 

Pancreatic  rennin  is  an  enzyme  found  in  the  gland  and  inthe  juice  which  coagu- 
lates neutral  or  alkaline  milk  (Ki^hne  and  Roberts  and  others).  This  enzyme  is 
not  identical  with  trypsin,  and  the  optimimi  of  its  action  lies  according  to  Vernon 
between  60°  and  65°.  According  to  Halliburton  and  Brodie  ^  casein  is  con- 
verted by  the  pancreatic  juice  of  the  dog  into  "pancreatic  casein,"  a  sub- 
stance which,  in  regard  to  solubility,  stands  to  a  certain  extent  between  casein 
and  paracasein  (see  Chapter  XIV),  and  which  is  converted  into  paracasein  by 
rennin.  Further  investigations  on  the  action  of  this  enzyme  upon  milk  and 
especially  upon  pure  casein  solutions  are  verj'  desirable 

The  property  of  pancreatic  juice  of  giving  plastein  precipitates  is  just  as 
inexplicable  as  in  the  case  of  the  gastric  juice  and  other  enzyme  solutions. 

Pancreatic  Calculi.  The  concrement  from  a  cystic  enlargement  ofWiRsuNG's 
duct  in  a  man,  as  analyzed  by  Baldoni,^  contained  in  1000  parts  as  follows: 
Water  34.4,  ash  126.7,  protein  substances  34.9,  free  fatty  acids  133,  neutral  fats 
124,  cholesterin  70.9,  soaps  and  pigment  499.1,  parts. 

Besides  the  enzymes  which  have  been  discussed  in  connection  with  the 
pancreatic  juice,  the  gland  also  contains  others,  among  which  can  be  men- 
tioned the  enzyme  which,  according  to  Stoklasa  and  his  collaborators, 
occurs  chieflv  in  organs  and  tissues  and  which  decomposes  sugar  into  alcohol 
and  carl)on  dioxide,  like  zymase.  According  to  tSiMACEK,^  in  the  pancreas 
the  glycolysis  by  means  of  alcoholic  fermentation,  and  the  hydrolysis  of 
the  disaccharides,  arc  united  together  as  a  specific  action,  and  he  has 
obtained  precipitates  from  cell-free  press-fluid  with  alcohol  and  ether 
which  brought  on  both  actions  without  bacterial  action.  The  statements 
as  to  the  importance  of  the  pancreas  for  glycolysis  are  very  contradictory, 
and  we  therefore  refer  the  reader  to  what  has  been  previously  stated  on 
this  subject  in  Chapter  VIII,  pages  302  and  303. 

*  Ki'ihne  and  Roberts,  Maly's  Jahresber.,  9;   see  also  Edkins,  Journ.  of  Physiol.,  12 
(literature  references);   Halliburton  and  Brodie,  ibid.,  20;   Vernon,  ibid.,  27. 
^  Maly's  Jahresber.,  29,  353. 
'  Stoklasa,  see  foot-note  1,  p.  303;  Simacek,  Centralbl.  f.  Physiol.,  17. 


CHEMICAL  PROCESSES   IN   THE   INTESTINE.  397 


V.    The  Chemical  Processes  in  the  Intestine. 

The  action  which  belongs  to  each  digestive  secretion  may  be  essen- 
tially changed  under  certain  conditions  by  being  mixed  with  other  digestive 
fluids  for  various  reasons,  and  also  by  the  action  of  the  enzymes  upon  each 
other;  ^  and  since  the  digestive  fluids  which  flow  into  the  intestine  are 
mixed  with  still  another  fluid,  the  bile,  it  will  be  readily  understood  that 
the  combined  action  of  all  these  fluids  in  the  intestine  makes  the  chemical 
processes  going  on  therein  very  complicated. 

As  the  acid  of  the  gastric  juice  acts  destructively  on  ptyalin,  this  enzyme 
has  no  further  diastatic  action,  even  after  the  acid  of  the  gastric  juice  has 
been  neutralized  in  the  intestine.  The  bile  has,  at  least  in  certain  animals, 
a  slight  diastatic  action,  which  in  itself  can  hardly  be  of  any  great  impor- 
tance, but  which  shows  that  the  bile  has  not  a  preventive  but  rather  a 
beneficial  influence  on  the  energetic  diastatic  action  of  the  pancreatic 
juice.  Martin,  Williams,  Pawlow,  and  Bruno  ^  have  observed  a 
beneficial  action  of  the  bile  on  the  diastatic  action  of  the  pancreas  infusion. 
To  this  may  be  added  that  the  organized  ferments  which  occur  habitually 
in  the  intestine  and  sometimes  in  the  food  have  partly  a  diastatic  action 
and  partly  produce  a  lactic-acid  and  butyric-acid  fermentation.  The 
maltose,  which  is  formed  from  the  starch,  seems  to  be  converted  into  dextrose 
in  the  intestine.  Cane-sugar  is  inverted  in  the  intestine,  and,  at  least 
in  certain  animals,  also  lactose.^  There  does  not  seem  to  be  any  doubt 
that  cellulose,  especially  the  fine  and  tender  varieties,  is  in  part  dissolved 
in  the  intestine;  still  the  products  formed  thereby  are  not  well  known. 
That  cellulose  undergoes  a  fermentation  in  the  intestine  by  the  action  of 
micro-organisms,  producing  marsh-gas,  acetic  acid,  and  butyric  acid,  has 
been  especially  shown  by  Tappeiner;  still  it  is  not  known  to  what  extent 
the  cellulose  is  destroyed  in  this  way.*  The  extensive  experiments  of 
Ellenberger  and  his  collaborators,  and  especially  the  observations  of 
ScHEUNERT  upon  the  digestion  of  cellulose,  are  very'  important.  Scheu- 
NERT^  finds  that  the  alkaline  contents  of  the  csecum  of  the  horse,  pig,  and 

^  See  Wroblewski  and  collaborators,  Hofmeister's  Beitrage,  1. 

^Martin  and  Williams,  Proceed,  of  Roy.  Soc,  45  and  48;  Bruno,  foot-note  1, 
p.  389. 

3  See  foot-note   2,  p.  379. 

*  On  the  digestion  of  cellulose  see  Henneberg  and  Stohmann,  Zeitschr.  f.  Biologie> 
21,  613;  V.  Knieriem,  ibid.,  67;  Hofmeister,  Arch.  f.  wiss.  u.  prakt.  Thierheilkunde, 
11;  Weiske,  Zeitschr.  f.  Biologic,  22,  373;  Tappeiner,  ibid.,  20  and  24;  Mallevre, 
Pfliiger's  Arch.,  49;  Omeliansky,  Arch.  d.  scienc.  biol.  de  St.  P^tersbourg,  7;  E.  Miiller, 
Pfliiger's  Arch.,  83;  Lohrisch,  Zeitschr.  f.  physiol.  Chem.,  4"  (literature). 

°  Ellenberger,  Arch.  f.  (Anat.  u.)  Physiol.,  1906;  Scheunert,  Zeitschr.  f.  physiol. 
Chem.,  48. 


398  DIGESTION. 

rabbit  have  the  power  of  dissolving  cellulose  to  a  considerable  extent.  This 
power  increases  as  the  abundance  of  micro-organisms  increases  and  vice 
versa;  but  even  in  the  complete  absence  of  these  organisms  considerable 
quantities  of  cellulose  are  dissolved.  The  secretion  or  the  extract  of  the 
csecal  mucosa  or  the  csecal  glands  does  not  contain  a  cellulose-dissohdng 
enzyme,  and  the  solution  of  cellulose  in  the  caecum  seems  therefore  to  be 
entirely  connected  with  the  micro-organisms  or  their  products. 

The  bile  has,  as  shown  by  Moore  and  Rockwood  ^  and  then  especially 
by  PFLiJGER,  the  property  to  a  high  degree  of  dissolving  fatty  acids,  espe- 
cially oleic  acid,  which  itself  is  a  solvent  for  other  fatty  acids,  and  hence, 
as  will  be  seen  later,  it  is  of  great  importance  in  the  absorption  of  fat.  It 
is  also  of  great  importance  that  the  bile,  as  previously  stated,  not  only 
activates  the  steapsinogen,  but  that,  as  first  shown  by  Nencki  and  Rach- 
roRD,2  it  accelerates  the  fat-splitting  action  of  the  steapsin.  According 
to  V.  FuRTH  and  Schutz  ^  the  bile-salts  are  the  active  constituents  of  the 
bile  in  this  cleavage,  and  the  fatty  acids  set  free  can  combine  with  the  alkalies 
of  the  intestinal  and  pancreatic  juices  and  the  bile,  producing  soaps  which 
are  of  great  importance  in  the  emulsification  of  the  fats. 

If  to  a  soda  solution  of  about  1-.3  p.  m.  Na2C03  is  added  pure,  perfectly 
neutral  olive-oil  in  not  too  large  quantity,  a  transient  emulsion  is  obtained 
after  vigorous  shaking.  If,  on  the  contrary,  one  adds  to  the  same  quantity 
of  soda  solution  an  equal  amount  of  commercial  olive-oil  (which  always 
contains  free  fatty  acids),  the  vessel  need  only  be  turned  over  for  the  two 
liquids  to  mix,  and  immediately  there  appears  a  very  finely  divided  and  per- 
manent emulsion,  making  the  liquid  appear  like  milk.  The  free  fatt}'  acids 
of  the  commercial  oil,  which  is  always  somewhat  rancid,  combine  with  the 
alkali  to  form  soaps  which  act  to  emulsify  the  fats  (Brucke,  Gad,  Loewen- 
THAL  4).  This  emulsifying  action  of  the  fatty  acids  split  off  by  the  pan- 
creatic juice  is  undoubtedly  assisted  by  the  habitual  occurrence  of  free  fatty 
acids  in  the  food,  as  well  as  by  the  splitting  off  of  fatty  acids  from  the 
neutral  fats  in  the  stomach  (see  page  363). 

Bile  completely  prevents  peptic  zymolysis  in  artificial  digestion,  because 
it  retards  the  swelling  up  of  the  proteins.  The  passage  of  bile  into  the 
stomach  during  digestion,  on  the  contrary,  seems,  according  to  several 
investigators,  especially  Oddi  and  Dastre,^  to  have  no  disturbing  action 
on  gastric  digestion.     According  to  Boldireff,^  in  continuous  starvation, 

'  Proceedings  of  Roy.  Soc,  60,  and  Journ.  of  Physiol.,  21.  In  regard  to  Pfliiger's 
work  see  Absorption. 

^  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  20;    Rachford,  Journal  of  Physiol.,  12. 

3  Centralbl.  f.  Physiol.,  20. 

*  Brucke,  Wien.  Sitzungsber.,  61,  Abt.  2;  Gad,  Arch.  f.  (Anat.  u.)  Physiol.,  1878; 
Loewenthal,  ibid.,   1897. 

"  Oddi,  in  Centralbl.  f.  Physiol.,  1,  312;   Dastre,  Arch,  de  Physiol.  (5),  2,  316. 

'Centralbl.  f.  Physiol.,  18,  457. 


ACTION  OF  THE   BILE.  399 

on  feeding  fat  and  food  rich  in  fat.  as  well  as  after  large  amounts  of  acid, 
a  mixture  of  bile,  pancreatic  juice,  and  intestinal  juice  passes  readily  into  the 
stomach.  After  food  rich  in  fat,  which  retards  the  secretion  of  gastric 
juice  and  the  motilit}'  of  the  stomach,  a  digestion  due  to  this  alkaline 
mixture  may  take  place  in  the  stomach. 

Bile  itself  has  no  solvent  action  on  proteins  in  neutral  or  alkaline  reaction, 
but  still  it  may  exert  an  influence  on  protein  digestion  in  the  intestine.  The 
acid  contents  of  the  stomach,  containing  an  alnnidance  of  proteins,  give 
with  the  bile  a  precipitate  of  proteins  and  bile-acids.  This  precipitate 
carries  a  part  of  the  pepsin  with  it,  and  for  this  reason,  and  also  on  account 
of  the  partial  or  complete  neutralization  of  the  acid  of  the  gastric  juice 
by  the  alkali  of  the  bile  and  the  pancreatic  juice,  the  pepsin  digestion  cannot 
proceed  further  in  the  intestine.  On  the  contrarj^,  the  bile  does  not  disturb 
the  chgestion  of  proteins  by  the  pancreatic  juice  in  the  intestine.  The 
action  of  these  digestive  secretions,  as  above  stated,  is  not  disturbed  by 
the  bile,  not  even  by  the  faintly  acid  reaction  due  to  organic  acids;  but, 
on  the  contrary,  the  action  of  trypsin  is  accelerated  by  the  bile.  In  a  dog 
killed  while  digestion  is  going  on,  the  faintly  acid,  bile-containing  material 
of  the  intestine  shows  regularly  a  strong  digestive  action  on  proteins. 

The  precipitate  formed  on  the  meeting  of  the  acid  contents  of  the 
stomach  with  the  l^ilc  easily  rcdissolves  in  an  excess  of  bile  and  also  in  the 
NaCl  formed  in  the  neutralization  of  the  hydrochloric  acid  of  the  gastric 
juice.  This  may  take  place  even  under  faintly  acid  reaction.  Since  in 
man  the  excretor}-  ducts  of  the  bile  and  the  pancreatic  juice  open  near  one 
another,  in  consequence  of  which  the  acid  contents  of  the  stomach  are 
probably  immediately  in  great  part  neutralized  by  the  bile  as  soon  as  it 
enters,  it  is  doubtful  whether  a  precipitation  of  proteins  by  the  bile  occurs 
in  the  intestine. 

Besides  the  previously  mentioned  processes  caused  by  enzymes,  there 
are  others  of  a  different  nature  going  on  in  the  intestine,  namely,  the  fer- 
mentation and  putrefaction  processes  caused  by  micro-organisms.  These 
are  less  intense  in  the  upper  parts  of  the  intestine,  but  increase  in  intensity 
towards  the  lower  part  of  the  same,  and  decrease  in  the  large  intestine 
because  of  the  consumption  of  fermentable  material  and  by  the  removal 
of  water  by  absorption.  Fermentation  processes,  but  only  very  slight 
putrefaction,  occur  in  the  small  intestine  of  man.  Macfadyex,  M. 
Nencki,  and  N.  Sieber  ^  have  investigated  a  case  of  human  anus  praeter- 
naturalis, in  which  the  fistula  occurred  at  the  lower  end  of  the  ileum,  and 
they  were  able  to  investigate  the  contents  of  the  intestine  after  it  had  been 
exposed  to  the  action  of  the  mucous  membrane  of  the  entire  small  intestine. 
The  mass  was  yellow  or  yellowish  brown,  due  to  bilirubin,  and  had  an  acid 

'  Arch.  f.  exp.  Path.  u.  Pharm.,  28. 


400  DIGESTION. 

reaction  which,  on  a  mixed  but  chiefly  animal  diet,  calculated  as  acetic 
acid,  amounted  to  1  p.  m.  The  contents  were  nearly  odorless,  having  an 
em]3yreumatic  odor  recalling  that  of  volatile  fatty  acids,  and  only  seldom 
had  a  putrid  odor  resembling  that  of  indol.  The  essential  acid  present  was 
acetic  acid,  accompanied  by  fermentation  lactic  acid  and  paralactic  acid, 
volatile  fatty  acids,  succinic  acid,  and  bile-acids.  Coagulable  proteins, 
peptone,  mucin,  dextrin,  dextrose,  and  alcohol  were  present.  Leucine 
and  tyrosine  could  not  be  detected. 

According  to  the  above-mentioned  investigators,  the  proteins  are  only 
to  a  very  slight  extent,  if  at  all,  decomposed  by  the  microbes  in  the  small 
intestine  of  man.  The  organisms  present  in  the  small  intestine  preferably 
decompose  the  carbohydrates,  forming  ethyl  alcohol  and  the  above-men- 
tioned organic  acids. 

Further  investigations  of  Jakowsky  and  of  Ad.  Schmidt  ^  led  to  the 
same  result,  namely,  that  in  man  the  putrefaction  of  the  proteins  takes 
place  chiefly  in  the  large  intestine,  and  the  conditions  are  the  same  in  car- 
nivora.  In  these  latter  it  has  been  possible  to  follow  the  intestinal  diges- 
tion by  investigating  the  contents  of  the  various  parts  of  the  intestine  as 
well  as  by  forming  fistulas.  London  and  Suluia  produced  fistulac  in  dif- 
ferent dogs  in  the  duodenum,  jejunum,  and  ileum,  and  could  follow  the 
digestion  of  boiled  egg-white,  A  complete  destruction  of  the  same  took 
place,  so  that  99.7  per  cent  of  the  protein  was  dissolved  and  flowed  out  of 
the  fistula  at  the  ileum  (2-3  cm.  in  front  of  the  caecum).  The  intestinal 
contents  gave  the  biuret  reaction  very  faintly,  and  the  dissolved  substance 
seemed  to  have  been  transformed  into  end-products.  Maetzke,^  who 
carried  on  his  investigations  on  a  dog  with  a  fistula  at  the  lower  end  of  the 
ileum,  on  feeding  meat  never  found  a  putrid  or  faecal  odor  to  the  intestinal 
contents.  The  digestion  and  absorption  of  the  meat  as  well  as  of  the  carbo- 
hydrate was  also  nearly  complete.  Leucine  and  tyrosine  were  looked  for 
but  not  found,  and  the  absence  of  these  bodies  was  explained  by  the  fact 
that  they  were  absorbed. 

Because  of  the  absorption  it  is  also  difficult  to  state  the  extent  of  de- 
struction of  the  proteins  in  the  intestine.  Several  experimenters  who  have 
investigated  the  intestinal  contents  of  dogs  during  the  digestion  of  meat 
have  detected  amino-acids  such  as  leucine,  tyrosine,  lysine,  and  arginine 
(KuTSCHER  and  Seemann).  glutamic  and  aspartic  acids,  alanine  (London), 
and  polypeptides  not  giving  the  biuret  reaction  (Abderhalden^). 

>  Jakowsky,  Arch,  des  scienc.  biol.  de  St.  Petersbourg,  1;  Ad.  Schmidt,  Arch.  f. 
Verdauungskr.,  4. 

»  London  and  SuUma,  Zeitschr.  f.  physiol.  Chem.,  40;  Maetzke,  Beobachtungen 
an  Hunden  mit  Anus  prseternaturahs,  Inaug. -Dissert.  Breslau,  1905. 

'  Kutscher  and  Seemann,  Zeitschr.  f.  physiol.  Chem.,  34;  Abderhalden,  ibid.,  44; 
London,  ibid.,  47. 


DIGESTION   IN  THE   INTESTINE.  401 

The  digestion  and  absorption  of  proteins  in  the  stomach  and  small 
intestine  may  be  nearly  complete,  but  this  is  not  always  so.  In  experi- 
ments with  raw  egg-white  Loxdox  and  Sulima  reobtained  about  73  per 
cent  of  the  coagulable  protein  from  the  ileum  fistula,  and  in  the  entire 
intestine  from  the  pylorus  to  the  csecum  only  about  12  per  cent  of  the 
food  substance  was  absorbed.  Also  in  milk-feeding  a  considerable  part 
of  the  protein  passes  into  the  large  intestine  (Berlatzki  i). 

As  above  remarked,  ordinarily  no  putrefaction  takes  place  in  the 
small  intestine  but  occurs  generally  only  in  the  large  intestine.  This 
putrefaction  of  the  proteins  is  not  the  same  as  the  pancreatic  digestion. 
In  putrefaction  the  decomposition  goes  much  further  and  a  mixture  of 
products  is  obtained  which  have  become  known  through  the  labors  of 
nimierous  investigators,  especially  Nexcki,  Baumaxx^,  Brieger,  H.  and 
E.  Salkowski,  and  their  pupils.  The  products  which  are  formed  in  the 
putrefaction  of  proteins  are  (in  addition  to  proteoses,  peptones,  amino-acids, 
and  ammonia)  imlol,  skatol,  paracresol,  phenol,  phenylpropionic  add,  and 
phenylacetic  acid,  also  paraoxyphenylacetic  acid  and  hydroparocumaric 
acid  (besides  paracresol.  produced  in  the  putrefaction  of  tyrosine),  volatile 
fatty  acids,  carbon  dioxide,  hydrogen,  marsh-gas,  methylmercaptan,  and 
sulphuretted  hydrogen.  In  the  putrefaction  of  gelatine  neither  tjTOsine 
nor  indol  is  formed,  while  glycocoll  is  produced  instead. 

Among  these  products  of  decomposition  a  few  are  of  special  interest 
because  of  their  beha^-ior  within  the  organism  and  because  after  their 
absorption  the}'  pass  into  the  urine.  A  few,  such  as  the  oxyacids,  pass 
unchanged  into  the  urine.  Others,  such  as  phenols,  are  directly  trans- 
formed into  ethereal  sulphuric  acids  by  spithesis,  and  are  eliminated  as 
such  by  the  urine;  on  the  contrary,  others,  such  as  indol  and  skatol,  are 
only  converted  into  ethereal  sulphuric  acids  after  oxidation  (for  details  see 
Chapter  XV).  The  quantity  of  these  bodies  in  the  urine  varies  also  with 
the  extent  of  the  putrefactive  processes  in  the  intestine;  at  least  this  is 
true  for  the  ethereal  sulphuric  acids.  Their  quantity  increases  in  the  urine 
■uith  a  stronger  putrefaction,  and  the  reverse  takes  place,  nameh',  a  disap- 
pearance from  the  urine,  or  a  great  reduction  in  quantity,  as  Baumaxx, 
Harley  and  Goodbody  -  have  shown  b}^  experiments  on  dogs,  when  the 
intestine  was  disinfected  liy  various  agents. 

Among  the  above-mentioned  putrefactive  products  in  the  intestine  the 
two  following,  indol  and  skatol,  should  be  especially  noted. 


'  See  Biochem.  Centralbl.,  2. 

^Baumann,  Zeitsclir.  f.  physiol.  Chem.,  10;    Harley  and  Goodbody,  Brit.  Med. 
Joum.,  1899. 


402  DIGESTION. 

CH 

Indol,    C8H7N  =  C6H4  CH,      and     Skatol,     or     methyl-indol, 

\      / 
NH 

C.CH3 

^    X 
C9H9N  =  C6H4  ^CH,  are  two  bodies  which  stand  in  close  relationship 

\       / 
NH 

to  the  indigo  substances  and  are  formed  in  variable  quantities  from  pro- 
tein compounds  under  different  conditions.  Hence  they  occur  habitually 
in  the  human  intestinal  canal,  and,  after  oxidation  into  indoxyl  and  skat- 
oxyl  respectively,  pass,  at  least  partly,  into  the  urine  as  the  corresponding 
ethereal  sulphuric  acids  and  also  as  glucuronic  acids. 

These  two  bodies  have  been  prepared  synthetically  in  many  ways. 
Both  may  be  obtained  from  indigo  by  reducing  it  with  tin  and  hydro- 
chloric acid  and  heating  this  reduction  product  with  zinc-dust  (Baeyer  ^). 
Indol  may  be  formed  from  skatol  by  passing  it  through  a  red-hot  tube. 
Indol  suspended  in  water  is  in  part  oxidized  into  indigo-blue  by  ozone 
(Nencki  2). 

Indol  and  skatol  crystallize  in  shining  leaves,  and  their  melting-points 
are  52°  and  95°  C.  respectively.  Indol  has  a  peculiar  excrementitious 
odor,  w^hile  skatol  has  an  intense  fetid  odor  (skatol  obtained  from  indigo  is 
odorless).  Both  bodies  are  easily  volatilized  by  steam,  skatol  more  easily 
than  indol.  They  may  both  be  removed  from  the  watery  distillate  by 
ether.  Skatol  is  the  more  insoluble  of  the  two  in  boiling  water.  Both  are 
easily  soluble  in  alcohol  and  give  with  picric  acid  a  compound  crys- 
tallizing in  red  needles.  If  a  mixture  of  the  two  picrates  be  distilled  with 
ammonia,  they  both  pass  over  without  decomposition;  while  if  they  are 
distilled  with  caustic  soda,  the  indol  but  not  the  skatol  is  decomposed. 
The  watery  solution  of  indol  gives  with  fuming  nitric  acid  a  red  liquid  and 
then  a  red  precipitate  of  nitroso-indol  nitrate  (Nencki).  It  is  better  first 
to  add  two  or  three  drops  of  nitric  acid  and  then  a  2  per  cent  solution  of 
potassium  nitrite,  drop  by  drop  (Salkowski^).  Skatol  does  not  give  this 
reaction.  An  alcoholic  solution  of  indol  treated  with  hydrochloric  acid 
colors  a  pine  chip  cherry-red.  Skatol  does  not  give  this  reaction.  Indol 
gives  a  deep  reddish-violet  color  with  sodium  nitroprussidc  and  alkali 
(Legal's  reaction).     On  acidifying  with  hydrochloric  acid  or  acetic  acid 

'  Annal.  d.  Chem.  u.  Pharm.,  140,  and  Suppl.,  7,  56;  also  Ber.  d.  deutsch.  chem. 
Gesellsch.,   1. 

*  Ber.  d.  deutsch.  chem.  Gesellsch.,  8,  727,  and  ibid.,  722  and  1517. 

'  Zeitschr.  f.  physiol.  Chem.,  8,  447.  In  regard  to  newer  reactions  for  indol  and 
skatol,  see  Steensma,  ibid.,  47. 


GASES  OF  THE   INTESTINE.  403 

the  color  becomes  pure  blue.  Skatol  does  not  act  the  same.  The  alkaline 
solution  is  yellow  and  becomes  violet  on  acidifying  with  acetic  acid  and 
boiling.  Skatol  dissolves  in  concentrated  hydrochloric  acid  with  a  violet 
coloration.  On  warming  skatol  with  sulphuric  acid  a  beautiful  purple-red 
coloration  is  obtained  (Ciamiciax  and  Magnanini  ^). 

For  the  detection  of  indol  and  skatol  in,  and  their  preparation  from, 
excrement  and  putrefying  mixtures,  the  main  points  of  the  usual  method 
are  as  follows:  The  mixture  is  distilled  after  acidifying  with  acetic  acid; 
the  distillate  is  then  treated  with  alkali  (to  combine  with  any  phenols 
which  may  be  present)  and  again  distilled.  From  this  second  distillate  the 
two  bodies,  after  the  addition  of  hydrochloric  acid,  are  precipitated  by 
picric  acid.  The  picrate  precipitate  is  then  distilled  with  ammonia.  The 
two  bodies  are  obtained  from  the  distillate  by  repeated  shaking  with  ether 
and  evaporation  of  the  several  ethereal  extracts.  The  residue,  containing 
indol  and  skatol,  is  dissolved  in  a  very  small  quantity  of  absolute  alcohol 
and  treated  with  8-10  vols,  of  water.  Skatol  is  precipitated,  but  not  the 
indol.  The  further  treatment  necessary  for  their  separation  and  purifica- 
tion will  be  found  in  other  works.^ 

The  gases  which  are  produced  by  the  decomposition  processes  are  mixed 
in  the  intestinal  tract  with  the  atmospheric  air  swallowed  with  the  saliva 
and  food,  and  as  the  gas  developed  in  the  decomposition  of  different  foods 
varies,  so  the  mixture  of  gases  after  various  foods  should  have  a  dissimilar 
composition.  This  is  found  to  be  true.  Oxygen  is  found  only  in  very  faint 
traces  in  the  intestine;  this  may  be  accounted  for  in  part  by  the  formation 
of  reducing  substances  in  the  fermentation  processes  which  combine  with 
the  oxygen,  and  partly,  perhaps  chiefly,  to  a  diffusion  of  the  oxygen  through 
the  tissues  of  the  walls  of  the  intestine.  To  show  that  these  processes  take 
place  mainly  in  the  stomach  the  reader  is  referred  to  page  372,  on  the 
composition  of  the  gases  of  the  stomach.  Nitrogen  is  habitualh'  found  in 
the  intestine,  and  it  is  probably  due  chiefly  to  the  swallowed  air.  The 
carbon  dioxide  originates  partly  from  the  contents  of  the  stomach,  parti}' 
from  the  putrefaction  of  the  proteins,  partly  from  the  lactic-acid  and 
butyric-acid  fermentation  of  carbohydrates,  and  partly  from  the  setting 
free  of  carbon  dioxide  from  the  alkali  carbonates  of  the  pancreatic  and 
intestinal  juices  by  their  neutralization  through  the  hydrochloric  acid  of 
the  gastric  juice  and  by  organic  acids  formed  in  the  fermentation.  Hydro- 
gen occurs  ir  largest  quantities  after  a  milk  diet,  and  in  smallest  quantities 
after  a  purely  meat  diet.  This  gas  seems  to  be  formed  chiefly  in  the 
butyric-acid  fermentation  of  carbohydrates,  although  it  may  occur  in 
large  quantities  in  the  putrefaction  of  proteins  under  certain  circumstances. 

»  Ber.  d.  d.  chem.  Gesellsch.,  21,  1928. 

"  For  quantitative,  colorimetric  determinations  of  indol  in  fseces,  see  Einhorn  and 
Huebner,  Salkowski's  Festschrift,  Berlin,  1904. 


404  DIGESTION. 

There  is  no  doubt  that  the  methylmercaptan  and  sulphuretted  hydrogen 
which  occur  normally  in  the  intestine  originate  from  the  proteins.  The 
marsh-gas  undoubtedly  originates  in  the  putrefaction  of  proteins.  As 
proof  of  this  Ruge  ^  found  26.45  per  cent  marsh-gas  in  the  human  intestine 
after  a  meat  diet.  He  found  a  still  greater  quantity  of  this  gas  after  a 
vegetable  (leguminous)  diet;  this  coincides  with  the  observation  that 
marsh-gas  may  be  produced  by  a  fermentation  of  carbohydrates,  but 
especially  of  cellulose  (Tappeiner  2).  Such  an  origin  of  marsh-gas,  especially 
in  herbivora,  is  to  be  expected.  A  small  part  of  the  marsh-gas  and  carbon 
dioxide  may  also  arise  from  the  decomposition  of  lecithin  (Hasebroek^). 

Putrefaction  in  the  intestine  not  only  depends  upon  the  composition  of 
the  food,  but  also  upon  the  albuminous  secretions  and  the  bile.  Among 
the  constituents  of  bile  which  are  changed  or  decomposed  there  are  not  only 
the  pigments — the  bilirubin  yields  urobilin  and  a  brown  pigment — ^but 
also  the  bile-acids,  especially  taurocholic  acid.  Glycocholic  acid  is  more 
stable,  and  a  part  is  found  unchanged  in  the  excrement  of  certain  animals, 
while  taurocholic  acid  is  so  completely  decomposed  that  it  is  entirely 
absent  in  the  fseces.  In  the  foetus,  on  the  contrary,  in  whose  intestinal 
tract  no  putrefaction  processes  occur,  undecomposed  bile-acids  and  bile- 
pigments  are  found  in  the  contents  of  the  intestine.  The  transformation 
of  bilirubin  into  urobilin  does  not  occur,  as  previously  stated,  in  man  in 
the  small  but  in  the  large  intestine. 

As  under  normal  conditions  no  putrefaction,  or  at  least  none  worth 
mentioning,  occurs  in  the  small  intestine,  and  as  often  nearly  all  the  pro- 
tein of  the  food  is  absorbed,  it  follows  that  ordinarily  it  is  the  secretions 
and  cells  rich  in  protein  which  undergo  putrefaction.  That  the  secretions 
rich  in  proteins  are  destr03^d  in  putrefaction  in  the  intestine  follows  from 
the  fact  that  putrefaction  may  also  continue  during  complete  fasting. 
From  the  observations  of  Mxjller^  upon  Cetti  it  was  found  that  the 
elimination  of  indican  during  starvation  rapidly  decreased  and  after  the 
third  day  of  starvation  it  had  entirely  disappeared,  while  the  phenol  elimina- 
tion, which  at  first  decreased  so  that  it  was  nearly  minimum,  increased 
again  from  the  fifth  day  of  starvation,  and  on  the  eight  or  ninth  day  it 
was  three  to  seven  times  as  much  as  in  man  under  ordinary  circumstances. 
In  doo-s,  on  the  contrary',  the  elimination  of  indican  during  starvation  is 
considerable,  but  the  phenol  elimination  is  slight.  Among  the  secretions 
which  undergo  putrefaction  in  the  intestine,  the  pancreatic  juice,  which 
putrefies  most  readily,  takes  first  place. 

From  the  foregoing  facts  it  must  be  concluded  that  the  products  formed 
by  the  putrefaction  in  the  intestine  are  in  part  the  same  as  those  formed 


*  Wien.    Sitzungsber.,   44.  ^  Zeitsch.  f.  Biologie,  20  and  24. 

s  Zeitschr.  f.  physiol.  Chem.,  12.  ••  Berlin,  klin.  Wochenschr.,  1887. 


PUTREFACTION  IN  THE  INTESTINE.  405 

in  digestion.  The  putrefaction  may  be  of  benefit  to  the  organism  in  so 
far  as  the  formation  of  such  products  as  proteoses,  peptones,  and  perhaps 
also  certain  amino-acids  is  concerned.  The  question  has  indeed  been 
asked  (Pasteur),  is  digestion  possible  ^YitllOut  micro-organisms?  Nuttal 
and  Thierfelder  have  shown  that  guinea-pigs  removed  from  the  uterus 
of  the  mother  by  Caesarian  section  could  with  sterile  air  digest  well  and 
assimilate  sterile  food  (milk  or  crackers)  in  the  complete  absence  of  bac- 
teria in  the  intestine,  and  developed  normally  and  increased  in  weight. 
ScHOTTELius  ^  has  arrived  at  other  results  by  experiments  with  hens. 
The  chickens,  hatched  under  sterile  conditions,  kept  in  sterile  rooms  and 
fed  with  sterile  food,  had  continuous  hunger  and  ate  abundantly,  but 
soon  died,  in  about  the  same  time  as  a  starving  chicken.  On  mixing  with 
the  food  of  other  chickens,  at  the  proper  time,  a  variety  of  bacteria  from 
hen  faeces,  they  gained  weight  again  and  recovered. 

The  bacterial  action  in  the  intestinal  canal  is,  at  least  in  certain  cases, 
necessary,  and  it  acts  in  the  interest  of  the  organism.  This  action  ma}-,  by 
the  formation  of  further  cleavage  products,  involve  a  loss  of  valuable  mate- 
rial to  the  organism,  and  it  is  therefore  important  that  putrefaction  in  the 
intestine  be  kept  within  certain  limits.  If  an  animal  is  killed  while  diges- 
tion in  the  intestme  is  going  on,  the  contents  of  the  small  intestine  give 
out  a  peculiar  but  not  putrescent  odor.  Also  the  odor  of  the  contents  of 
the  large  intestine  is  far  less  offensive  than  a  putrefying  pancreas  infusion 
or  a  putrefying  mixture  rich  in  protein.  From  this  one  may  conclude  that 
putrefaction  in  the  intestine  is  ordinarily  not  nearly  so  intense  as  outside 
of  the  organism. 

It  seems  thus  to  be  provided,  under  physiological  conditions,  that 
putrefaction  shall  not  proceed  too  far,  and  the  factors  which  here  come 
mider  consideration  are  probably  of  different  kinds.  Absorption  is  un- 
doubtedly one  of  the  most  important  of  them,  and  it  has  been  proved  by 
actual  observ^ation  that  the  putrefaction  increases,  as  a  rule,  as  the  absorp- 
tion is  checked  and  fluid  masses  accumulate  in  the  intestine.  The  character 
of  the  food  also  has  an  unmistakable  influence,  and  it  seems  as  if  a  large 
quantity  of  carbohydrates  in  the  food  acts  against  putrefaction  (Hirsch- 
ler2).  It  has  been  shown  by  Pohl,  Bierxacki,  Rovighi,  Wixterxitz, 
ScHMiTZ,  and  others  ^  that  milk  and  kephir  have  a  specially  strong  pre- 
ventive action  on  putrefaction.  This  action  is  not  due  to  the  casein,  but 
chiefly  to  the  lactose  and  also  in  part  to  the  lactic  acid. 

1  Nuttal  and  Thierfelder,  Zeitsclir.  f.  physiol.  Chem.,  21  and  22;  Schottelius,  Arch 
f.  Hygiene,  34  and  42. 

^  Hirschler.,  Zeitsclir.  f.  phj^siol.  Chem.,  10;  Zimnitzki,  ibid.,  39  (literature). 

^  Schmitz,  ibid.,  17,  401,  which  gives  references  to  the  older  Uterature,  and  19.  See 
also  Salkowski,  Centralbl.  f.  d.  med.  Wiss.,  1893,  467,  and  Seelig.  Virchow's  Arch.,  146 
(literature). 


406  DIGESTION. 

A  specially  strong  preventive  action  on  putrefaction  has  been  ascribed 
for  a  long  time  to  the  bile.  This  anti-putrid  action  does  not  exist  in  neutral 
or  faintly  alkaline  bile,  which  itself  easily  putrefies,  but  to  the  free  bile- 
acids,  especially  taurocholic  acid  (Maly  and  Emich,  LixdbergerI).  There 
is  no  question  that  the  free  bile-acids  have  a  strong  preventive  action  on 
putrefaction  outside  of  the  organism,  and  it  is  therefore  difficult  to  deny 
such  an  action  in  the  intestine.  Notwithstanding  this  the  anti-putrid 
action  of  the  bile  in  the  intestine  is  not  considered  by  certain  investigators 
(VoiT,  RoHMANX,  HiRscHLER  and  Terra Y,  Landauer  and  Rosenberg  2) 
^s  of  great  importance. 

Biliary  fistulas  have  been  established  so  as  to  study  the  importance  of 
the  bile  in  digestion  (Schwann,  Bloxdlot,  Bidder  and  Schmidt,^  and 
others).  As  a  result  it  has  been  observed  that  with  fatty  foods  an  imper- 
fect absorption  of  fat  regularly  takes  place  and  the  excrements  contain, 
therefore,  an  excess  of  fat  and  have  a  light-gray  or  pale  color.  The  extent 
of  deviation  from  the  normal  after  the  operation  is  essentially  dependent 
upon  the  character  of  the  food.  If  an  animal  is  fed  on  meat  and  fat,  then 
the  quantity  of  food  must  be  consideral^ly  increased  after  the  operation, 
otherwise  the  animal  will  become  very  thin,  and  indeed  die  with  symptoms 
of  starvation.  In  these  cases  the  excrements  have  the  odor  of  carrion,  and 
this  was  considered  a  proof  of  the  action  of  the  bile  in  checking  putrefac- 
tion. The  emaciation  and  the  increased  want  of  food  depend,  naturally, 
upon  the  imperfect  al^sorption  of  the  fats,  whose  high  calorific  value  is 
reduced  and  must  be  replaced  by  the  taking  up  of  larger  quantities  of  other 
nutritive  bodies.  If  the  quantity  of  proteins  and  fats  be  increased,  then 
the  latter,  which  can  be  only  very  incompletely  absorbed,  accumulate 
in  the  intestine.  This  accumulation  of  the  fats  in  the  intestine  only 
renders  the  action  of  the  digestive  juices  on  proteins  more  difficult, 
and  thus  increases  the  amount  of  putrefaction.  This  explains  the  ap- 
pearance of  fetid  faeces,  whose  pale  color  is  not  due  to  a  lack  of  bile- 
pigments,  but  to  a  surplus  of  fat  (Rohmann,  Voit).  If  the  animal  is, 
on  the  contrary,  fed  on  meat  and  carbohydrates,  it  may  remain  quite 
normal,  and  the  leading  off  of  the  bile  does  not  cause  any  increased  putre- 
faction. The  carbohydrates  may  be  uninterrupedly  absorbed  in  such 
large  quantities  that  they  replace  the  fat  of  the  food,  and  this  is  the  reason 
why  the  animal  on  such  a  diet  does  not  become  emaciated.  As  with  this 
diet  the  putrefaction  in  the  intestine  is  no  greater  than  under  normal  con- 

*  Maly  and  Emich,  Monatshefte  f.  Chem.,  4;  Lindberger,  foot-note  2,  p.  393. 

^  Voit,  Beitr.  ziir  Biologie,  Jubilaumschrift,  Stuttgart,  1882;  Rohmann,  Pfliiger's 
Arch.,  29;  Hirschler  and  Terray,  Maly's  Jahresber.,  20;  Landauer,  Math.  u.  Naturw. 
Ber.  aus  Ungarn,  15;  Rosenberg,  Arch.  f.  (Anat.  u.)  Physiol.,  1901. 

^  Schwann,  Midler's  Arch.  f.  Anat.  u.  Physiol.,  1844;  Blondlot,  cited  from  Bidder 
and  Schmidt,  Verdauungssafte,  etc.,  98. 


PUTREFACTION   IX  THE  INTESTINE.  407 

ditions  even  though  the  bile  is  absent,  it  would  seem  that  the  bile  in  the 
intestine  exercises  no  preventive  action  on  putrefaction. 

To  this  conclusion  the  objection  may  be  made  that  the  carboh3''drates, 
which  are  capable  of  checking  putrefaction,  can,  so  to  speak,  undertake 
the  anti-putrid  action  of  the  bile.  But  as  there  are  also  cases  (in  dogs 
with  biliary  fistula)  where  the  intestinal  putrefaction  is  not  increased  with 
exclusive  meat  diet,^  it  is  maintained  that  the  absence  of  bile  in  the  intes- 
tine, even  by  exclusive  carbohydrate  food,  does  not  always  cause  an  in- 
creased putrefaction. 

Although  the  question  as  to  the  manner  in  which  the  putrefactive 
processes  in  the  intestine  under  physiological  conditions  are  kept  within 
certain  limits  cannot  be  answered  positively,  still  it  may  be  asserted  that 
the  acid  reaction  of  the  upper  parts  of  the  intestine,  and  the  absorption  of 
water  in  the  lower  parts,  are  important  factors. 

That  the  acid  reaction  in  the  intestine  has  a  preventive  influence  on  the 
putrefactive  processes  follows  from  the  existing  relation  between  the  degree 
of  acidity  of  the  gastric  juice  and  the  putrefaction  in  the  intestine.  After 
the  investigations  and  observations  of  Kast,  Stadelmaxn,  Wasbutzki, 
BiERNACKi  and  ]\Iester  had  proved  that  an  increased  putrefaction  in  the 
intestine  occurred  when  the  quantitj^  of  hydrochloric  acid  in  the  gastric 
juice  was  diminished  or  deficient.  Schmitz  2  has  lately  shown  in  man  that 
on  the  administration  of  hydrochloric  acid,  producing  a  hj-peracidity  of  the 
gastric  juice,  the  putrefaction  in  the  intestine  may  be  checked.  The  ques- 
tion arises  whether  the  reaction  in  the  small  intestine  is  alwa3's  acid  and 
whether  the  acidity  is  strong  enough  to  prevent  putrefaction.  In  this 
connection  it  must  be  recalled  that  the  acidity  of  the  contents  of  the  small 
intestine  is  not  due  to  hydrochloric  acid,  but  chiefly  to  organic  acids,  acid 
salts,  and  free  carbon  dioxide.  There  are  several  statements  as  to  the  reac- 
tion of  the  intestinal  contents,  although  they  are  somewhat  contradictory, 
by  Moore  and  Rock  wood,  Moore  and  Bergin,  ]\Iatthes  and  Mar- 
QUARDSEX,  I.  MuxK,  Nexcki  and  Zaleski,  Hemmeter.3  From  these 
statements  one  can  conclude  that  the  reaction  may  vaiy  not  only  among 
different  animals,  but  also  in  the  same  animals  under  different  conditions. 
There  is  no  doubt  that  the  acid  reaction  in  many  cases  is  due  to  the  presence 
of  organic  acids.  On  testing  with  various  indicators  it  has  been  shown 
that  sometimes  the  upper  parts,  and  often  the  lower  parts,  are  acid,  due 
to  acid  salts  such  as  XaHC03  and  free  COo,  and  finally  that  in  certain 

*  See  Hirschler  and  Terray ,  1.  c. 

^  Zeitsclir.  f.  physiol.  Chem.,  19,  401,  which  includes  all  the  pertinent  literature. 

^  Moore  and  Rockwood,  Journ.  of  Physiol.,  21;  IMoore  and  Bergin,  Amer.  Journ. 
of  Physiol.,  3;  Matthes  and  Marquardsen,  Maly's  Jahresber.,  28;  ]*Iunk,  Centralbl.  f. 
Physiol.,  16;  Nencki  and  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  2";  Hemmeter,  Pfl;,ger's 
Arch.,  81. 


408  DIGESTION. 

animals  the  intestinal  contents  are  alkaline  throughout.  The  question 
how,  under  these  conditions,  putrefaction  is  excluded,  cannot  be  explained. 
It  is  possible,  as  Bienstock  admits,  that  the  explanation  lies  in  an  antag- 
onistic bacterial  action  and  that  the  carbohydrates,  especially  lactose, 
which  retard  putrefaction,  form  a  good  nutritive  media  for  those  bacteria 
which  destroy  the  putrefactive  producers  or  retard  their  development. 
Perhaps  also,  according  to  the  experience  of  Conradi  and  Kurpjuweit,^ 
the  autotoxines  produced  by  the  intestinal  bacteria  may,  by  their  antiseptic 
action,  keep  the  putrefactive  processes  in  the  intestine  within  bounds. 

Excrements.  It  is  evident  that  the  residue  which  remains  after  com- 
plete digestion  and  absorption  in  the  intestine  must  be  different,  both 
qualitatively  and  quantitatively,  according  to  the  variety  and  quantity  of 
the  food.  In  man  the  quantity  of  excrement  from  a  mixed  diet  is  120-150 
grams,  with  30-37  grams  of  solids,  per  twenty-four  hours,  while  the  quantity 
from  a  vegetable  diet,  according  to  Voit,^  was  333  grams,  with  75  grams 
of  solids.  With  a  strictly  meat  diet  the  excrements  are  scanty,  pitch-like, 
and  black.  The  scanty  excrements  in  starvation  have  a  similar  appear- 
ance. A  large  quantity  of  coarse  bread  yields  a  great  amount  of  light- 
colored  excrement.  In  these  cases  the  faeces  are  also  habitually  poorer 
in  nitrogen  than  after  food  rich  in  protein.  The  individuality  also  plays 
an  important  role  in  the  utility  of  the  food  and  the  formation  of  faeces 
(ScHiERBECK  ^).  If  there  is  a  large  proportion  of  fat,  it  takes  a  lighter, 
clay-like  appearance.  The  decomposition  products  of  the  bile-pigments 
seem  to  play  only  a  small  part  in  the  normal  color  of  the  f  seces. 

The  constituents  of  the  faeces  arc  of  different  kinds.  In  the  excrements 
are  found  digestible  or  absorbable  constituents  of  the  food,  such  as  muscle 
fibres,  connective  tissues,  lumps  of  casein,  grains  of  starch,  and  fat,  which 
have  not  had  sufficient  time  to  be  completely  digested  or  absorbed  in  the 
intestinal  tract.  In  addition  the  excrements  contain  indigestible  bodies, 
such  as  the  remains  of  plants,  keratin  substances,  and  others;  also  form- 
elements  originating  from  the  mucous  coat  and  the  glands;  constituents 
of  the  different  secretions,  such  as  mucin,  cholic  acid,  dyslysine,  and 
cholesterin  (koprosterin  or  stercorin),  purine  bases,*  and  enzymes;  mineral 
bodies  of  the  food  and  the  secretions;  and,  lastly,  products  of  putrefac- 
tion or  of  digestion,  such  as  skatol,  indol,  volatile  fatty  acids,  purine 
bases,  lime,  and  magnesia  soaps.     Occasionally,  also,  parasites  of  different 

*  Bienstock,  Arch.  f.  Hygiene,  39;  Conradi  and  Kurpjuweit,  Munch,  med.  Wochen- 
schr.,  1905. 

2  Zeitschr.  f.  Biologie,  25,  264. 
^  Arch.  f.  Hygiene,  51. 

*  In  regard  to  the  purine  bases  in  feces,  see  Hall,  Journ.  of  Path,  and  Bacteriol.,  9; 
Schittenhelm,  Arch.  f.  kiln.  Med.,  SI;  Schittenhehn  and  Kriiger,  Zeitschr.  f.  physiol. 
Chem.,  45. 


EXCREMENTS.  409 

kinds  occur;  and  lastly,  the  excrements  contain  micro-organisms  of  various 
species. 

That  the  mucous  membrane  of  the  intestine  by  its  secretion  and  by 
the  abundant  quantity  of  detached  epithelium  contributes  essentially  to 
the  formation  of  excrement  follows  from  the  discovery  first  made  by 
L.  HeRxMaxx  and  substantiated  by  others/  that  a  clean,  isolated  loop  of 
intestine  collects  material  similar  to  faeces.  Human  faeces  seem  to  consist 
in  greater  part  of  intestinal  secretion  and  only  in  a  smaller  part  of  residue 
from  food  on  a  meat  or  milk  diet.  Many  foods  produce  a  large  quantity 
of  faeces  chiefly  by  causing  an  abundant  secretion.^ 

The  reaction  of  the  excrements  is  very  variable,  but  in  man  with  a 
mixed  diet  it  is  neutral  or  faintly  alkaline.  It  is  often  acid  in  the  inner 
part,  while  the  outer  layers  in  contact  with  the  mucous  coat  have  an  alka- 
line reaction.  In  nursing  infants  it  is  habitually  acid.  The  odor  is  perhaps 
chiefly  due  to  skatol,  which  was  first  found  in  the  excrements  by  Brieger, 
and  so  named  b}'  him.  Indol  and  other  substances  also  take  part  in  the 
production  of  odor.  The  color  is  ordinarily  light  or  dark  brown,  and 
depends  above  all  upon  the  nature  of  the  food.  Medicinal  bodies  may 
give  the  faeces  an  abnormal  color.  The  excrements  are  colored  black  b}' 
bismuth,  yellow  by  rhubarij,  and  green  by  calomel.  This  last-mentioned 
color  was  formerly  accounted  for  by  the  formation  of  a  little  mercur}- 
sulphide,  but  now  it  is  said  that  calomel  checks  the  putrefaction  and  the 
decomposition  of  the  bile-pigments,  so  that  a  part  of  the  bile-pigments 
passes  into  the  faeces  as  biliverdin.  In  the  yolk-yellow  or  greenish-yellow 
excrements  of  nursing  infants  one  can  detect  bilirubin.  Neither  bilirubin 
nor  Ijiliverdin  seems  to  exist  in  the  excrements  of  mature  persons  under 
normal  conditions.  On  the  contrary,  there  is  found  stercohilin  (Masius 
anel  Vanlair),  which  is  identical  with  urobilin  (Jaffe  ^).  Bilirubin  may 
occur  in  pathological  cases  in  the  faeces  of  mature  persons.  It  has  been 
observeel  in  a  crystallized  state  (as  haematoidin)  in  the  faeces  of  children 
as  well  as  of  grown  persons. 

The  absence  of  bile  (acholic  faeces)  causes  the  excrements  to  have,  as 
above  stated,  a  gray  color,  due  to  large  quantities  of  fat;  this  may,  however, 
be  partly  attributed  to  the  absence  of  bile-pigments.  In  these  cases  a 
large  quantity  of  crystals  has  been  observed  which  consist  chiefly  of  mag- 
nesium soaps  or  sodium  soaps.     Hemorrhage  in  the   upper  parts  of  the 

"Hermann,  Pfli'iger's  Arch.,  46.  See  also  Ehrenthal,  ibid.,  48;  Berenstein,  ibid., 
53;  Klecki,  Centralbl.  f.  Physiol.,  7;  736,  and  F.  Voit,  Zeitschr.  f.  Biologie,  29;  v. 
Moraczewski,  Zeitschr.  f.  physiol.  Cheni.,  25. 

^  In  regard  to  the  constitution  of  faeces  with  various  foods,  see  Hammerl,  Kermauner, 
Moeller,  and  Prausnitz,  Zeitsclir.  f.  Biologie,  35,  and  Poda,  Micko,  Prausnitz  and 
Miiller,  ibid.,  39. 

^  See  bile-pigments,  Chapter  VIII,  and  vu-obilin,  Cliapter  XV. 


410  DIGESTION. 

digestive  tract  yields,  when  it  is  not  very  abundant,  a  dark-brown  excre- 
ment, due  to  hsematin. 

ExcRETiN,  so  named  by  Marcet,'  is  a  crystalline  body  occurring  in  human 
excrement,  but  which,  according  to  Hoppe-Seyler,  is  perhaps  only  impure 
cholesterin  (koprosterin  or  stercorin?).  Excretolic  acid  is  the  name  given  by 
Marcet  to  an  oily  body  with  an  excrementitious  odor. 

In  consideration  of  the  very  variable  composition  of  excrements,  their 
quantitative  analyses  are  of  little  value  and  therefore  will  be  omitted.^ 

Meconium  is  a  dark  brownish-green,  pitchy,  mostly  acid  mass  without 
any  strong  odor.  It  contains  greenish-colored  epithelium  cells,  cell-detritus, 
numerous  fat-globules,  and  cholesterin  plates.  The  amount  of  water 
is  720-800,  and  solids  280-200  p.  m.  Among  the  solids  there  are  mucin, 
bile-pigments,  and  bile-acids,  cholesterin,  fat,  soaps,  traces  of  enz3^mes, 
calcium  and  magnesium  phosphates.  Sugar  and  lactic  acid,  soluble 
protein  bodies  and  peptones,  also  leucine  and  tyrosine  and  the  other  pro- 
ducts of  putrefaction .  occurring  in  the  intestine,  are  absent.  Meconium 
may  contain  imdecomposed  taurocholic  acid,  bilirubin  and  biliverdin,  but 
it  does  not  contain  any  stercobiline,  which  is  considered  as  proof  of  the 
non-existence  of  putrefactive  processes  in  the  digestive  tract  of  the  foetus. 

In  medico-legal  cases  it  is  sometimes  necessary  to  decide  whether  spots 
on  linen  or  other  substances  are  caused  l^y  meconium.  In  such  cases  the 
following  conditions  exist:  The  spot  caused  by  meconium  has  a  brownish- 
green  color  and  can  be  easily  separated  from  the  material  because,  on 
account  of  the  ropy  property  of  the  meconium,  it  is  difficult  to  wet  through. 
When  moistened  with  water  it  does  not  develop  any  special  odor,  but  on 
warming  with  dilute  sulphuric  acid  it  smells  somewhat  fetid.  It  forms 
with  water  a  slimy,  greenish-yellow  liquid  containing  brown  flakes.  The 
solution  gives  with  an  excess  of  acetic  acid  an  insoluble  precipitate  of 
mucin;  on  boiling  it  does  not  coagulate.  The  filtered,  watery  extract 
responds  to  Gmelin's,  but  still  better  to  Huppert's  reaction  for  bile- 
pigments.  The  liquid  precipitated  by  an  excess  of  milk  of  lime  gives  a 
nearly  colorless  filtrate,  which  after  concentration  shows  Pettenkofer's 
reaction. 

The  contents  of  the  intestine  under  abnormal  conditions  are  perhaps  less  the 
subject  of  chemical  analysis  than  of  an  inspection  and  microscopical  investiga- 
tion or  bacteriological  examination.  On  this  account  the  question  as  to  the 
properties  of  the  contents  of  the  intestine  in  different  diseases  cannot  be  thor- 
oughly treated  here.^ 

*  Annal.  de  chim.  et  de  phys.,  59. 

^  In  regard  to  these  analyses  as  well  as  to  the  faeces  under  abnormal  conditions 
and  to  the  pertinent  literature,  see  Ad.  Schmidt  and  J.  Strassburger,  Die  Fgeces  des 
Menschen,  etc.,  Berlin,  1901  and  1902. 

'  See  Schmidt  and  Strassburger,  1.  c. 


INTESTINAL  CONCRKMENTS.  411 

Appendix. 

JNTESTINAL    CONCREMENTS. 

Calculi  occur  xevy  seldom- in  the  human  intestine  or  in  the  Litestine  of 
camivora,  but  they  are  quite  common  in  herbivora.  Foreign  bodies  or 
undigested  residues  of  food  may,  when  for  some  reason  or  other  they  are 
retained  in  the  intestine  for  some  time,  become  incrusted  with  salts,  espe- 
cially ammonium-magnesium  phosphate  or  magnesimn  phosphate,  and 
these  salts  usually  form  the  chief  constituent  of  the  concrements.  In  man 
they  are  sometimes  oval  or  round,  yellow,  yellowish  gray,  or  brownish  gray, 
of  variable  size,  consisting  of  concentric  layers  and  containing  chiefly 
ammonium-magnesium  phosphate  and  calcimn  phosphate,  besides  a  small 
quantity  of  fat  or  pigment.  The  nucleus  ordinaril}-  consists  of  some  foreign 
body,  such  as  the  stone  of  a  fruit,  a  fragment  of  bone,  or  something  similar. 
In  those  countries  where  bread  made  from  oat -bran  is  an  important  food, 
we  often  find  in  the  large  intestine  balls  similar  to  the  so-called  hair-balls 
(see  below).  Such  calculi  contain  calciiun  and  magnesium  phosphate 
(about  70  per  cent),  oat-bran  (15-18  per  cent),  soaps  and  fat  (about  10 
per  cent).  Concretions  which  contain  ven,^  much  fat  (about  74  per  cent) 
occaiiionally  occur,  and  those  consisting  of  fibrin  clots,  sinews,  or  pieces  of 
meat  incrusted  with  phosphates  are  also  rare. 

Intestinal  calculi  often  occur  in  animals,  especially  in  horses  fed  on 
bran.  These  calculi,  which  attain  a  very  large  size,  are  hard  and  hea^y 
(as  much  as  8  kilos)  and  consist  in  great  part  of  concentric  layers  of  ammo- 
nium-magnesium phosphate.  Another  variety  of  concrements  which 
occurs  in  horses  and  cattle  consists  of  gray-colored,  often  verj-  large,  but 
relatively  light  stones  which  contain  plant  residues  and  earthy  phosphates. 
Stones  of  a  third  variety  are  sometimes  cylindrical,  sometimes  spherical, 
smooth,  shining,  brownish  on  the  surface,  consisting  of  matted  hairs  and 
plant-fibres,  and  termed  hair-balls.  The  so-called  ".egageopiL/E.''  which 
probably  originate  from  the  axtilopus  rupicapra,  belong  to  this  group, 
and  are  generally  considered  as  nothing  else  than  the  hair-balls  of  cattle. 

The  so-called  oriental  bezoar-stone  belongs  also  to  the  intestinal  concre- 
ments, and  probably  originates  from  the  intestinal  tract  of  the  capra 
^GAGRUS  and  axtilope  dorcas.  There  may  exist  two  varieties  of  bezoar- 
stones.  One  is  olive-green,  faintly  shining  and  formed  of  concentric  layers. 
On  heating  it  melts  with  the  development  of  an  aromatic  odor.  It  con- 
tains as  chief  constituent  lithofellic  acid,  C20H36O4,  which  is  related  to 
cholic  acid,  and  besides  this  a  bile-acid,  lithobilic  acid.  The  others  are 
nearly  blackish  brown  or  dark  green,  ven,'  glossy,  consisting  of  concentric 
layers,  and  do  not  melt  on  heating.    They  contain  as  chief  constituent 


412  DIGESTION. 

ellagic  acid,  a  derivative  of  gallic  acid,  of  the  formula  CuHgOg,  which, 
according  to  Graebe,i  is  the  dilactone  of  hexaoxydiphenyldicarboxylic  acid 
and  which  gives  a  deep-blue  color  with  an  alcoholic  solution  of  ferric  chlo- 
ride. This  last-mentioned  bezoar-stone  originates,  to  all  appearances, 
from  the  food  of  the  animal. 

Ambergris  is  general!}'  considered  an  intestinal  concrement  of  the  sperm- 
whale.  Its  chief  constituent  is  ambrain,  which  is  a  non-nitrogenous  substance 
perhaps  related  to  cholesterin.  Ambrain  is  insoluble  in  water  and  is  not  changed 
by  boihng  alkalies.     It  dissolves  in  alcohol,  ether,  and  oils. 


VI.    Absorption. 

The  problem  of  digestion  consists  in  part  in  separating  the  valuable  con- 
stituents of  the  food  from  the  useless  ones  and  dissolving  or  transforming 
them  into  forms  which  are  adapted  for  the  processes  of  absorption.  In 
discussing  the  absorption  processes  we  must  treat  of  the  form  into  which 
the  different  foods  are  changed  before  absorption,  of  the  manner  in 
which  this  is  accomplished,  and,  lastly,  of  the  forces  which  act  in  these 
processes. 

Before  we  can  answer  the  question  as  to  the  form  in  which  the  proteins 
are  absorbed  from  the  intestinal  canal,  it  is  of  interest  to  leam  whether  the 
animal  body  can,  perhaps,  also  utilize  such  protein  a^  is  introduced  intra- 
venously, subcutaneously,  or  into  a  body-cavity,  i.e.,  evading  the  intestinal 
canal,  or,  as  Oppexheimer  calls  it.  parenteral. 

Since  the  first  investigations  of  Zuxtz  and  v.  Merixg  on  this  subject 
several  expsrimenters,  such  as  Neumeister.  Friedexth.u.  and  Lew^ax- 
DowsKY,  MuxK  and  Lewaxdowsky,  Oppexheimer.  Mexdel  and  Rock- 
wood,  and  others,^  have  sho^ATi,  without  any  doubt,  that  the  animal  body 
can  more  or  less  completely  utilize  different,  parenterally  introduced  pro- 
teins, although  different  varieties  of  animals  show  a  difference  in  this  regard. 
Still  we  do  not  know  where  and  how  these  foreign  proteins  are  changed 
and  assimilated. 

If  the  animal  body  can  assimilate  parenterally  introduced  protein,  then 
the  question  arises,  whether  it  can  also  take  up  undigested  protein  from  the 
intestinal  canal  and  utilize  it.  In  this  regard  we  have  the  observations  of  a 
large  number  of  investigators,  such  as  Brucke,  Bauer  and  Voit.  Eich- 


»  Ber.  d.  d.  chem.  Gesellsch.,  3(5. 

^  Zuntz  and  v.  Me:ing,  Pfliiger's  Arch.,  32;  Neumeister,  Verh.  d.  phys.-med. 
Gesellsch.  zu  W  rzburg,  1889,  and  Zeitschr.  f.  Biologie,  2";  Friedenthal  and  Lewan- 
dowsky,  Arch.  f.  (Anat.  u.)  Physiol.,  1899;  Munk  and  Lewandowsky,  ibid.,  1899,  Suppl.; 
Oppenheimer,  Hofmeister's  Beitrage,  i;  Mendel  and  Rockwood,  Amer.  Joum.  of 
Physiol.,  12. 


ABSORPTION   OF  PROTEINS.  413 

HORST,  CzERXY  and  Latschexberger,  ^'oIT  and  Friedlaxder,^  who 
have  shown  that  non-pcptonized  protein  can  be  absorbed  from  the  intestine. 
In  the  experiments  of  the  two  last -mentioned  investigators  neither  casein 
(as  milk)  nor  hydrochloric-acid  myosin  or  acid  albuminate  (in  acid 
solution)  was  absorbed,  while,  on  the  contrary,  about  21  per  cent  of  oval- 
bumin or  seralbumin  and  69  per  cent  of  alkali  albuminate  (dissolved  in 
alkali)  were  absorbed.  Mexdel  and  Rockwood,  on  the  contrary,  in 
experiments  with  casein  and  edestin  in  the  living  intestinal  loop,  could 
prove  only  the  slightest  absorption  on  excluding  digestion  as  completely 
as  possible,  while  the  corresponding  proteoses  were  abundantly  absorbed. 

It  is  difficult  to  decide  in  these  experiments  as  to  how  far  the  proteins 
were  taken  up  in  an  actually  unchangeel  or  partly  modified  form.  The 
alimentary  albuminuria  obsen-ed  repeatedly  after  the  introdtiction  of 
large  quantities  of  protein  into  the  intestinal  canal  speaks  for  an  absorption 
of  undigesteel  protein  uneler  certain  circumstances.  To  deciele  this  question 
the  biological  method,  using  the  precipitine  reaction,  has  l^een  made  use 
of,  and  AscoLi  and  ^'igxo.^  using  this  method,  claim  to  have  shown  the 
passage  of  non-mocUfied  protein  into  the  blood  and  lymph.  Based  upon 
many  investigations  on  this  subject  we  can  consider  it  possilile  that  under 
certain  circiunstances,  as  on  flooding  the  intestmal  canal  with  protein, 
with  a  greater  permeability  of  the  intestinal  wall,  as  in  new-born  and 
sucking  animals,  and  with  a  diminished  modification  by  the  gastric  juice, 
a  passage  of  non-modifieel  protein  may  take  place  in  the  blood-vessels,  but 
that  under  normal  conditions  this  is  not  the  case,  or  at  least  does  not  take 
place  to  any  mentionable  degree.  As  a  rule,  the  absorption  of  protein 
follows  a  modification  of  the  same,  and  the  next  question  is  whether  the 
proteins  are  chiefly  absorbed  as  proteoses  or  peptones  or  as  simpler  atomic 
complexes. 

This  question  cannot  be  answered  for  the  present.  Investigations  on 
the  con+^nts  of  the  stomach  anel  intestine  have  shoT\7i  the  presence  of 
proteoses  and  peptones  as  well  as  non-biuret-giving  atomic  complexes  and 
amino-acids.  The  results  of  the  investigations  of  Schmidt-Mulheim. 
Ellexberger  and  Hofmeister,  Ewald  and  Gumlich.  Zuxz.  Reach, 
Kutscher  and  Seemaxx,  Abderhaldex.  Glaessxeb,  and  others  3  have 

*  Briicke,  '\\'ien.  Sitzungsber.,  a9;  Bauer  and  Voit,  Zeitschr.  f.  Biologic,  o;  Eich- 
horst,  Pfliiger's  Arch.,  4;  Czerny  and  Latschenberger,  Virchow's  Arch.,  59;  Voit  and 
Friedlander,  Zeitschr.  f.  Biologic .  33. 

2  Zeitschr.  f.  physiol.  Chem.,  39. 

^Schmidt-Midheim.  Arch.  f.  (Anat.  u.)  Physiol.,  1879;  Ellenberger  and  Hofmeister, 
ibid.,  1890;  Ewald  and  Gumlich,  Berlm.  klin.  Wochensclir.,  1S90;  E.  Zunz,  Hof- 
meister's  Beitriige,  3;  Reach,  ibid.,  4;  Zmiz,  Aimal.  de  la  soc.  roy.  d.  scienc.  de  Bru- 
xelles,  13;  Kutscher  and  Seemann,  Zeitsclir.  f.  physiol.  Chem.,  34  and  35;  Abderhalden, 
ibid.,  44;  Glaessner,  Zeitsclir.  f.  klin.  Med.,  52. 


414  DIGESTION. 

given,  as  was  to  be  expected,  contradictory  and  variable  results,  and 
as  the  absorption  runs  more  or  less  parallel  with  digestion  the  quantities 
of  the  various  products  found  in  the  intestinal  canal  cannot  give  any  positive 
conclusions  as  to  the  amounts  produced. 

The  proteoses,  as  well  as  the  peptones,  have  been  repeatedly  found  in 
the  stomach  and  the  intestine,  and  therefore  the  question  has  been  raised 
for  a  long  tims  how  these  bodies  are  absorbed  and  how  they  are  introduced 
in  the  tissues.  The  generally  accepted  view  is  that  they  do  not  pass  into 
the  blood  through  the  lymphatics,  but  through  the  intestinal  epithelium, 
and  this  view  is  based  essentially  on  the  two  following  conditions:  On 
completely  isolating  the  chyle  from  the  blood  circulation,  the  protein 
absorption  from  the  intestine  is  not  impaired  (Ludwig  and  Schmidt- 
MtJLHEiM) ;  and  on  a  diet  rich  in  protein  the  quantity  thereof  in  the  chyle 
(in  man)  was  not  noticeal^ly  increased  (Munk  and  Rosensteix).  Asher 
and  Barbera  ^  have  shown  in  experiments  on  a  clog  that  the  quantity  of 
protein  in  the  lymph  was  slightly  increased  after  partaking  of  considerable 
protein.  This  experiment  does  not  disprove  the  assertion  of  a\IuxK  that 
the  blood-vessels  form  nearly  the  exclusive  exit  of  the  proteins  from  the 
intestinal  tract. 

After  a  diet  rich  in  proteins  neither  proteoses  nor  peptones  are  found 
in  the  blood  or  the  chyle.  Nor  are  they  ^^resent  in  the  urine;  and  the 
absence  of  these  bodies  in  the  blood  after  digestion  cannot  be  explained  by 
the  statement  that  they,  like  the  proteoses  (peptones)  injected  subcutane- 
ously  or  directly  into  the  blood,  are  quickly  eliminated  through  the  kidneys 
(Plosz  and  Gyergyai,  Hofmeister,  Schmidt-Mulheim^).  It  might  be 
supposed  that  the  proteoses  (peptones)  formed  in  digestion  are  retained  by 
the  liver,  and  that  this  is  the  reason  why  they  are  not  found  in  the  blood. 
This  explanation  does  not  seem  to  be  sufficient.  Neumeister  has  inves- 
tigated the  portal  blood  of  rabbits  into  whose  stomachs  large  quantities 
of  proteoses  and  peptones  had  been  introduced,  without  finding  traces  of 
the  bodies  in  question. 

He  has  also  shown  that  when  the  liver  of  a  dog  is  supplied  with 
portal  blood  to  which  peptone  is  added  (ampho-peptone),  this  is  not 
retained  by  the  liver.  Shore  has  arrived  at  similar  results  in  regard  to 
the  importance  of  the  liver,  and  has  also  shown  that  the  spleen  cannot 
transform  peptone.  Peptone  seems  to  pass  neither  into  the  blood  nor 
the  chylous  vessels,   and  the  following  ol:>servation  of  Ludavig  and  Sal- 


'  Sclimid1>Miilheim,  Arch.  f.  (Anat.  u.)  Physiol.,  1877;  Munk  and  Rosenstein,  Vir- 
chow's  Arch,,  123;  Asher  and  Barbera,  Centralbl.  f.  Physiol.,  11,  403;  Munk,  ibid., 
11,  585.     See  also  Mendel,  Amer.  Journ.  Physiol.,  2. 

'  Plosz  and  Gyergyai,  Pfliiger's  Arch.,  10;  Hofmeister,  Zeitschr.  f.  physiol.  Chem., 
5;   Sclimidt-M  ilheim,  Arch.  f.  (Anat.  u.)  Phy.siol.,  1880. 


ABSORPTION  OF    PROTEINS.  415 

viOLi  bears  out  this  assumption.  These  investigators  introduced  a  peptone 
solution  into  a  double-Ugatured,  isolated  piece  of  the  small  intestine,  which 
was  kept  alive  by  passing  defibrinated  blood  through  it,  and  observed  that 
the  peptone  disappeared  from  the  intestme,  but  that  the  blood  passing 
through  did  not  contain  any  peptone,  Cathcart  and  Leathes  ^  with  their 
own  experiments  as  a  basis,  give  another  interpretation  of  Salvioli's  obser- 
vations, namely,  by  the  statement  that  the  disappearance  of  the  peptone 
from  the  loop  of  the  intestine  depends  upon  a  hydrolysis  of  the  same.  On 
the  other  hand,  they  also  found  that  no  peptone  was  taken  up  by  the 
circulating  blood. 

It  must  be  remarked  in  connection  with  this  view  that,  according  to 
Embden  and  Knoop,  and  Langstein,  proteoses  sometimes  occur  in  blood- 
serum,  and  also  that  Nolf  ^  has  found,  after  abundant  absorption  of 
proteoses  from  the  intestine,  a  small  amount  in  the  blood.  This  occurrence 
of  proteoses  in  the  blood  is  not  contradictory  to  the  view  that  the  chief 
quantity  of  proteoses  and  peptones  does  not  pass  from  the  intestine  into 
the  blood  as  such. 

Many  observations  indicate  that  the  proteoses  and  peptones  are  trans- 
formed in  some  way  in  the  intestine  or  intestinal  wall,  and  a  retransforma- 
tion  of  proteoses  into  protein  is  considered  most  plausible. 

Certain  investigators,  such  as  v.  Ott,  Nadine  Popoff,  and  Julia 
Brinck,^  are  of  the  opinion  that  the  proteoses  and  peptones  are  transformed 
into  seralbumin  before  they  pass  into  the  walls  of  the  digestive  tract.  This 
transformation  is  brought  about  by  means  of  the  epithelium-cells,  as  also 
by  the  vital  activity  of  a  fungus  called  by  Julia  Brinck  Micrococcus 
restituens.     No  positive  proofs  have  been  presented  to  support  this  view. 

The  view  that  the  transformation  of  the  proteoses  and  peptones  takes 
place  after  they  have  been  taken  up  by  the  mucous  membrane  has  better 
foundation.  According  to  the  observations  of  Hofmeister,'*  the  walls 
of  the  stomach  and  the  intestine  are  the  only  parts  of  the  body  in  which 
proteoses  (peptones)  occur  constantly  during  digestion,  and  the  fact  that 
proteoses  (peptones)  at  the  temperature  of  the  body  disappeared  after  a 
time  from  the  excised  but  apparently  still  living  mucous  coat  of  the  stom- 
ach, also  confirm  this. 

This  disappearance  of  proteoses  is  considered  by  Hofmeister  as  a 
transformation  into  ordinary  protein.     For  such  a  transformation  of  pro- 

'  Neumeister,  Sitzungsber.  d.  phys.-med.  Gesellsch.  zu  Wiirzburg,  1889,  and  Zeitsclir. 
f.  Biologic,  24;  Shore,  Journ.  of  Physiol.,  11;  Salvioli,  Arch.  f.  (Anat.  u.  (Physiol.. 
1880,  Suppl.;  Cathcart  and  Leathes,  Journ.  of  Physiol.,  33. 

^  See  Chapter  VI,  foot-note  1,  p.  183. 

^  V.  Ott,  Arch.  f.  (Anat.  u.)  Physiol.,  1883;  Popoff,  Zeitschr.  f.  Biologic,  25;  Brinck, 
ibid.,  453. 

*  Zeitschr.  f.  physiol.  Chem.,  6,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  19,  20,  and  22. 


416  DIGESTION. 

teoses  in  the  mucosa  of  the  stomach,  Glaessner  i  has  suggested  new 
experimental  evidence,  while  the  Hofmeister  school  (Embden  and  Knoop) 
consider  the  regeneration  of  peptone  into  coagulable  protein  in  the  intes- 
tine as  not  proved. 

According  to  Hofmeister  the  leucocytes,  which  are  increased  during 
digestion,  play  an  important  part  in  the  transformation  of  the  proteoses 
and  peptones.  They  may  in  the  first  place  take  up  the  proteoses  (pep- 
tones) and  be  the  means  of  transporting  them  to  the  blood,  and  secondly 
by  their  growth,  regeneration,  and  increase  may  stand  in  close  relationship 
to  the  transformation  and  assimilation  of  the  bodies.  Heidenhain,  who 
considers  that  the  transformation  of  peptones  into  protein  in  the  mucous 
membrane  is  positively  settled,  does  not  attribute  so  great  an  importance 
to  the  leucoc3rtes  in  the  absorption  of  the  peptones,  chiefly  on  the  groimd 
of  comparative  estimation  of  the  quantity  of  absorbed  peptones  and  leuco- 
cytes. He  considers  it  as  more  probable  that  the  reconversion  of  the 
peptones  into  protein  takes  place  in  the  epithelium  layers.  This  view  is 
further  corroborated  by  the  investigations  of  Shore.^ 

On  account  of  the  discovery  of  erepsin  by  CoHxXHeim,  the  theorv^  as  to 
the  absorption  of  proteins  has  taken  another  direction.  There  seems  to 
be  a  tendency  to  lean  towards  the  view  that  the  proteoses  and  peptones 
are  split  in  the  intestine,  or  in  the  intestinal  mucosa,  into  simpler  bodies 
which  do  not  give  the  biuret  test  and  from  which  the  proteins  are  regen- 
erated. The  question  whether  the  active  agent  is  erepsin  or  trypsin 
is  only  of  secondary  importance,  as  both  of  these  enzymes  split  the  pro- 
teoses and  peptones  alike. 

According  to  the  investigations  of  the  Hofmeister  school  on  pepsin 
digestion,  and  of  Fischer  and  Abderhalden  on  tr^^psin  digestion  (see 
Chapter  II),  the  disappearance  of  the  biuret  test  does  not  indicate  a  com- 
plete cleavage  of  the  proteins  into  amino-acids,  since  peptoids  or  poly- 
peptides occur;  consequently  it  is  for  the  present  not  possible  to  say  to  what 
extent  the  proteins  are  broken  dowTi  in  the  intestinal  canal,  and  how  far  the 
amino-acids  and  more  complex  atomic  groups  not  giving  the  biuret  reaction 
are  produced.  It  is  just  as  difficult  to  state  with  positiveness,  although 
feeding  experiments  with  this  in  view  have  been  carried  out,  how  far  a 
regeneration  of  protein  from  such  abiuret  peptides  or  from  amino-acids 
is  possible. 

The  possibility  of  keeping  an  animal  for  a  certain  time  in  nitrogenous 
equilibrium  with  abiuret  digestion  products  was  first  demonstrated  by  Loewi. 
He  fed  dogs  with  an  abiuret  digestion  mixture  of  pancreas  tissue  and  kept 
them  in  nitrogenous  equilibrium  for  more  than  a  month.     Henderson 

*  Hofmeister's  Beitrage,  1. 

^  Heidenhain,  Pfluger's  Arch.,  43;   Shore,  1.  c. 


ABSORPTION  OF   PROTEINS.  417 

and  Dean  were  also  able  in  a  bitch  to  observe  nitrogenous  equilibrium 
for  at  least  a  few  days  by  feeding  the  abiuret  products  of  the  acid  cleavage 
of  meat,  while  Lesser,  on  the  contrary,  could  not  bring  the  animal  in 
nitrogenous  equilibrium  by  using  fibrin  digested  with  trypsin.  These 
negative  results  not  only  confront  the  positive  results  of  Loewi  but  also 
the  observations  of  Abderhalden  and  Rona,  as  well  as  of  Hexriques 
and  Hansen,!  and  there  is  no  doubt  that  mice,  rats,  and  dogs  can  be  kept 
for  at  least  a  certain  time  in  nitrogenous  equilibrium  with  abiuret  digestion 
products  consisting  in  great  part  of  monamino-acids.  Of  special  interest 
is,  no  doubt,  the  fact  that  in  the  experiments  of  Abderhalden  and  Rona, 
as  well  as  of  Henriques  and  Hansen,  the  abiuret  products  obtained  from 
casein  by  pancreatic  digestion  could  protect  the  animal  from  loss  of  nitro- 
gen, while  the  products  obtained  by  acid  hydrolysis  of  casein  or  a  mixture 
of  amino-acide  corresponding  to  casein  (Abderhalden  and  Rona)  could 
not  do  this.  Remarkable  is  the  observ^ation  of  Henriques  and  Hansen 
that  the  products  (monamino-acids?)  not  precipitable  by  phosphotungstic 
acid  could  also  cover  the  nitrogen  loss.  It  is  hardly  possible  to  draw  any 
positive  conclusions  from  the  above  experiments  as  to  the  ability  of  the 
animal  body  to  regenerate  proteins  by  synthesis  from  abiuret  digestion 
products.  It  is  just  as  difficult  to  say  whether  and  to  what  extent  a 
synthesis  of  protein  from  the  simple  cleavage  products  takes  place  in  the 
intestinal  wall.  Cathcart  and  Leathes  found  that  when  peptone  or 
end-products  of  pancreatic  digestion  were  absorbed  by  the  intestinal  loop 
the  amount  of  nitrogenous  substances  in  the  blood  not  precipitated  by 
tannic  acid  regularly  increased,  which  seems  to  indicate  that  these  simple 
cleavage  products  were  taken  up  by  the  blood. 

The  extent  of  the  protein  absorption  is  dependent  essentially  upon  the 
kind  of  food  introduced,  since  as  a  rule  the  protein  substances  from  an 
animal  source  are  much  more  completely  absorbed  than  from  a  vegetable 
source.  As  proof  of  this  the  following  observations  are  given:  In  his  experi- 
ments on  the  utilization  of  certain  foods  in  the  intestinal  canal  of  man  Rub- 
ner  found  that  with  an  exclusively  animal  diet,  on  partaking  of  an  average 
of  738-884  grams  of  fried  meat  or  948  grams  of  eggs  per  day,  the  nitrogen 
deficit  with  the  excrement  was  only  2.5-2.8  per  cent  of  the  total  nitrogen  in- 
troduced. With  a  strictly  milk  diet  the  results  were  somewhat  unfavorable, 
since  after  partaking  of  4100  grams  of  milk  the  nitrogen  deficit  increased 
to  12  per  cent.  The  conditions  are  quite  different  with  vegetable  food,  as 
shown  by  the  researches  of  Meyer,  Rubner,  Hultgren  and  Lander- 
gren,  who  made  experiments  with  various  kinds  of  rj^e  bread  and  found 

'  Loewi,  Arch.  f.  exp.  Path.  u.  Pharm.,  4S.  See  also  Henderson  and  Dean,  Amer. 
Journ.  of  Physiol.,  9;  Lesser,  Zeitschr. .  f .  Biologie,  45;  Abderhalden  and  Rona, 
Zeitschr.  f.  physiol.  Chem.,  42,  44,  and  47;  Henriques  and  Hansen,  ibid.,  43. 


418  DIGESTION. 

that  the  loss  of  nitrogen  through  the  faeces  amounted  to  22-48  per  cent. 
Experiments  with  other  vegetable  foods,  and  also  the  investigations  of 
Schuster,  Cra:\ier,  Meinert,  Mori.^  and  others  on  the  utilization  of  foods 
with  mixed  diets,  have  led  to  similar  results.  With  the  exception  of  rice, 
wheat  bread,  and  certain  verj^  finely  divided  vegetable  foods,  it  is  found 
in  general  that  the  nitrogen  deficit  by  the  faeces  increases  with  a  larger 
quantity  of  vegetable  material  in  the  food. 

The  reason  for  this  is  manifold.  The  large  quantity  of  cellulose  fre- 
quently present  in  vegetable  foods  impedes  the  absorption  of  proteins. 
The  greater  irritation  produced  by  the  vegetable  food  itself  or  by  the  organic 
acids  formed  in  the  fermentation  in  the  intestinal  canal  causes  a  more 
violent  peristalsis,  which  drives  the  contents  of  the  intestine  faster  than 
otherwise  along  the  intestinal  canal.  Another  and  most  important  reason. 
is  the  fact  that  a  part  of  the  vegetable  protein  substances  seem  to  be 
indigestible. 

In  speaking  of  the  functions  of  the  stomach  we  stated  that  after  the 
removal  or  excision  of  this  organ,  an  abundant  digestion  and  absorption 
of  proteins  may  take  place.  It  is  therefore  of  interest  to  learn  how  the 
digestion  and  absorption  of  proteins  go  on  after  the  extirpation  of  the 
second  protein-digesting  organ,  the  pancreas.  In  this  connection  there 
are  the  observations  on  animals  after  complete  or  partial  extirpation  of 
the  gland  by  Minkowski  and  Abelmann,  Sandmeyer,  V.  Harley,  after 
destroying  the  gland  by  Rosenberg,  and  also  in  man  after  closing  the 
pancreatic  duct  by  Harley  and  Deucher.^  In  all  these  different  cases  such 
discrepant  figures  have  been  obtained  for  the  utilization  of  the  proteins — 
between  80  per  cent  after  the  apparently  complete  exclusion  of  pancreatic 
juice  in  man  (Deucher)  and  IS  per  cent  after  extirpation  of  the  gland  in 
dogs  (Harley) — that  one  can  hardly  draw  any  clear  conception  as  to  the 
extent  and  importance  of  the  trypsin  digestion  in  the  intestine.  This  is 
not  to  be  wondered  at,  because  one  would  expect  that  in  such  cases  the 
other  digestive  fluids  undergo  variation,  and  indeed  to  various  degrees  in 
the  different  cases.  Zunz  and  Mayer  3  have  also  found  that  in  dogs  (meat 
digestion)  the  tying  of  the  pancreatic  passages  is  essentially  compensated 
for  by  an  increased  secretion  of  pepsin  and  other  proteolytic  enzymes,  and 

» Rubner,  Zeitsclir.  f.  Biologie,  15;  Meyer,  ibid.,  7;  Hultgren  and  Landergren, 
Nord.  med.  Arch.,  21;  Schuster,  in  Voit's  "Untersuch.  d.  Kost,"  etc.,  142;  Cramer, 
Zeitschr.  f.  physiol.  Chem.,  6;  Meinert,  "tjber  Massennahrung,"  Berlin,  1885;  Kell- 
ner  and  Mori,  Zeitschr.  f.  Biologie,  25. 

2  Abelmann,  "Uber  die  Ausnitzung  der  Nahrungsstoffe  nach  Pankreasexstirpa- 
tion"  (Inaug.-Dissert.  Dorpat,  1890),  cited  from  Maly's  Jahresber.,  20;  Sandmeyer, 
Zeitschr.  f.  Biologie,  31;  Rosenberg,  Pfl:  ger's  Arch.,  70;  Harley,  Joum.  of  Pathol, 
and  Bacteriol.,  1895;  Deucher,  Correspond.  Blatt  f.  Schweiz.  Aerzte,  28. 

^  Mem.  de  I'Acad.  roy.  de  medic,  de  Belg.,  18. 


ABSORPTION  OF  CARBOHYDRATES.  419 

that  in  this  case  the  demolition  of  the  protein  in  the  stomach  goes  further 
than  in  a  normal  animal. 

The  carbohydrates  are,  it  seems,  chiefl}'  absorbed  as  monosaccharides. 
Dextrose,  levulose,  and  galactose  are  probably  absorbed  as  such.  The 
two  disaccharides,  saccharose  and  maltose,  ordinarily  undergo  an  inversion 
in  the  intestinal  tract  and  are  converted  into  dextrose  and  levulose.  Lactose 
is  also,  at  least  in  certain  animals,  inverted  in  the  intestine.  In  other 
mature  animals,  on  the  contrary,  if  the  lactase  formation  is  not  excited  by 
milk  food,  the  sugar  is  not  inverted  or  only  to  a  slight  extent  (Voit  and 
LusK,  Weinland,  PoRTiER,  RoHMANN  and  Nagano),  and  it  probably  is  ab- 
sorbed as  such  in  these  animals  if  it  does  not  undergo  fermentation,  or,  as 
RoHMANX  and  Nagano  ^  assumed,  if  it  is  not  transformed  in  the  intes- 
tinal mucosa  in  some  unknown  way.  An  absorption  of  non-inverted  car- 
bohydrates is  not  improbable,  and  according  to  Otto  and  v.  Merixg^  the 
portal  blood  contains  besides  dextrose  a  dextrin-like  carbohydrate  after 
a  carbohydrate  diet.  A  part  of  the  carbohydrates  is  destroyed  by  fermen- 
tation in  the  intestine,  with  the  formation  of  lactic  and  acetic  acids  and 
other  bodies. 

The  different  varieties  of  sugars  are  absorbed  with  var^dng  degrees  of 
rapidity,  but  as  a  general  thing  absorption  occurs  very  quickly.  This  ab- 
sorption takes  place  more  quickly  in  the  upper  part  of  the  intestine  than  in 
the  lower  part  (Roh^l^xx,  Laxxois  and  Lepixe,  Rohmaxx  and  Nagaxo^). 
It  is  generally  admitted  that  the  simpler  sugars  are  more  quickly  absorbed 
than  the  disaccharides,  while  the  statements  as  to  the  absorption  of  the 
disaccharides  differ  somewhat  (Hedox,  Albertoxi,  Waymouth  Reid, 
Rohmaxx  and  Nagaxo).  There  seems  to  be  no  doubt  that  lactose  is 
absorbed  more  slowly  than  the  two  other  disaccharides.  According  to 
the  extensive  experiments  of  Rohmaxx  and  Nagaxo,  saccharose  is 
absorbed  more  quickly  than  maltose.  Nagaxo  '^  contends  that  the  pentoses 
are  absorbed  more  slowly  than  hexoses. 

On  the  introduction  of  starch  even  in  very  considerable  quantities  into 
the  intestinal  tract  no  dextrose  passes  into  the  urine,  a  condition  which 
probably  depends  in  thi?  case  upon  the  absorption  and  assimilation  and  the 
slow  saccharification  taking  place  simultaneously.  If,  on  the  contrary, 
large  quantities  of  sugar  are  introduced  at  one  time,  then  an  elimination  of 
sugar  b}'  the  urine  takes  place,  and  this    elimination   of    sugar   is    called 


*  Voit  and  Liisk,  Zeitschr.  f.  Biologie,  2S;    Rolimann  and  Nagano,  Pfliiger's  Arch.. 
95,  which  contains  the  references  to  the  Hterature. 

^  Otto,  see  Maly's  Jahresber.,  17;    v.  Mering,    Arch.  f.  (Anat.  u.)  Physiol.,  1877. 
^  Lannois  et  Lepine,  Arch,  de  Physiol.  (3),  1;    RShmann,  Pfliiger's  Arch.,  41;    sea 
also  foot-note  1. 

*  In  regard  to  the  Hterature  on  the  absorj^tion  of  sugars,  see  foot-note  1. 


420  DIGESTION. 

alimentary  glycosuria.  In  these  cases  the  assimilation  of  the  sugar  and  the 
absorption  do  not  occur  at  the  same  time,  hence  the  liver  and  the  remaining 
organs  do  not  have  the  necessary  time  to  fix  and  utilize  the  sugar.  This 
glycosuria  may  also  in  part  be  due  to  the  fact  that  the  introduction  of 
considerable  quantities  of  sugar  forces  this  substance  to  be  absorbed  not 
only  in  the  ordinary  way  through  the  blood-vessels  to  the  liver  (see  below), 
but  also  in  part  by  passing  into  the  blood  circulation  through  the 
lymphatic  vessels,  thus  evading  the  liver. 

That  quantity  of  sugar  to  which  we  must  raise  the  ingested  substance  in 
order  to  produce  an  alimentary  glycosuria  gives,  according  to  Hofmeister,i 
the  assimilation  limit  for  that  same  sugar.  This  limit  is  different  for  various 
kinds  of  sugar;  and  it  also  varies  for  the  same  sugar  not  only  in  different 
animals,  but  also  in  chfferent  members  of  the  same  species,  as  also  in  the 
same  individual  under  different  circumstances.  In  general  it  can  be  said 
that  in  regard  to  the  ordinary  varieties  of  sugar,  such  as  dextrose,  levulose, 
saccharose,  maltose,  and  lactose,  the  assimilation  hmit  is  highest  for  dex- 
trose and  lowest  for  lactose.  It  must  be  admitted  that  with  an  over- 
abundant quantity  of  sugars  in  the  intestinal  tract  the  disaccharides  do 
not  have  sufficient  time  for  their  complete  inversion,  and  this  has  been 
directly  shown  by  Rohmann  and  Nagano.  It  is,  therefore,  not  remarkable 
that  also  disaccharides  have  been  found  in  the  urine  in  cases  of  alimentary 
glycosuria.2 

The  investigations  of  Ludwig  and  v.  Mering  and  others  have  explained 
how  the  sugars  enter  into  the  blood-stream,  namely,  that  they  as  well 
as  other  bodies  soluble  in  water  do  not  ordinarily  pass  over  into  the 
chylous  vessels  in  measurable  quantities,  but  are  chiefly  taken  up  by  the 
blood  in  the  capillaries  of  the  villi  and  in  this  way  pass  into  the  mass  of 
the  Ijlood.  These  investigations  have  been  confirmed  by  observations  of 
I.  MuNK  and  Rosenstein  ^  on  human  l^eings. 

The  reason  why  the  sugars  and  other  soluble  bodies  do  not  pass  over 
into  the  chylous  vessels  in  appreciable  quantity  is,  according  to  Heiden- 
HAIN,4  to  be  found  in  the  anatomical  conditions,  in  the  arrangement  of  the 
capillaries  close  under  the  layer  of  cpitheUum.  Ordinarily  these  capillaries 
find  the  necessary  time  for  the  removal  of  the  water  and  the  solids  dis- 
solved in  it.     But  when  a  large  quantity  of  liquid,  such  as  a  sugar  solution. 


>  Arch.  f.  exp.  Path.  u.  Pharm.,  25  and  26. 

^  For  the  Uterature  in  regard  to  the  passage  of  various  kinds  of  sugars  into  the  urine, 
see  C.  Voit,  Ueber  die  Glykogenbildung,  Zeitschr.  f .  Biologic,  28,  and  F.  Voit,  foot-note 
3,  p.  293.  See  also  Bkimenthal,  Zur  Lehre  von  der  Assimilationsgrenze  der  Zucker- 
arten,  Inaug.-Dissert.  1903,  Strassburg. 

'v.  Mering,  Arch.  f.  (Anat.  u.)  Physiol.,  1877;  Munk  and  Rosenstein,  Virchow's 
Arch.,  123. 

*  Pfhjger's  Arch.,  43,  Suppl. 


ABSORPTIOiN    OF    CARBOHYDRATES.  421 

is  introduced  into  the  intestine  at  once,  this  is  not  possible,  and  in  these 
cases  a  part  of  the  dissolved  bodies  passes  into  the  chylous  vessels  and  the 
thoracic  duct  (Ginsberg  and  Rohmann  i). 

The  introduction  of  larger  quantities  of  sugar  into  the  intestine  at  one 
time  can  readily  cause  a  disturbance  with  diarrhceal  evacuations  of  the 
intestine.  If  the  carbohydrate  is  introduced  in  the  form  of  starch,  then 
very  large  quantities  may  be  absorbed  without  causing  any  disturbance, 
and  the  absorption  may  be  very  complete.  Rubner  found  the  following: 
On  partaking  508-670  grams  of  carbohydrates,  as  wheat  bread,  per  day 
the  part  not  absorbed  amounted  to  only  0.8-2.6  per  cent.  For  peas,  where 
357-588  grams  were  eaten,  the  loss  was  3.6-7  per  cent,  and  for  potatoes 
(718  grams)  7.6  per  cent.  Constantinidi  found  on  partaking  367-380 
grams  of  carbohydrates,  chiefly  as  potatoes,  a  loss  of  only  0.4-0.7  per  cent. 
In  the  experiments  of  Rubner,  as  also  of  HuLTGREisr  and  Landergren,^ 
with  rye  bread  the  utilization  of  carbohydrates  was  less  complete,  and 
the  loss  in  a  few  c-ases  rose  even  to  10.4-10.9  per  cent.  It  at  least  follows 
from  the  experiments  made  thus  far  that  man  can  absorb  more  than  500 
grams  of  carl^ohydrates  per  diem  without  difficulty. 

We  generally  consider  the  pancreas  as  the  most  important  organ  in 
the  digestion  and  absorption  of  amylaceous  bodies,  and  it  is  a  question  how 
these  ])odies  are  al^sorbed  after  the  extirpation  of  the  pancreas.  As  on  the 
absorption  of  proteins,  so  also  on  the  absorption  of  starch,  the  observations 
have  given  variable  results.  In  certain  cases  the  absorption  was  not 
impaired,  while  in  others  it  was,  on  the  contrary,  rather  diminished,  and 
with  dogs  devoid  of  pancreas  it  has  been  found  that  the  absorption  was 
decreased  to  50  percent  of  the  starch  partaken  (Rosenberg,  Cavazzani^). 

Emulsification  used  to  be  considered  as  of  the  greatest  importance  in 
the  absorption  of  fats,  and  this  emulsion  occurs  in  the  chyle  on  the  intro- 
duction into  the  intestine  of  not  only  neutral  fats,  but  also  of  fatty  acids. 
The  fatty  acids  do  not  exist  as  such  in  the  emulsified  fat  of  the  chyle.  The 
investigations  of  I.  Munk,  later  confirmed  by  others,  have  shown  that  the 
fatty  acids  undergo  in  great  part  a  synthesis  into  neutral  fats  in  the  walls  of 
the  intestine,  and  are  carried  as  such  by  the  stream  of  chyle  into  the  blood 
This  synthesis  seems  to  take  place  in  the  mucous  membrane  (Moore). 
The  experimental  evidence  thus  far  obtained  for  this  assumption  is  not 
very  conclusive.^ 

^  Ginsberg,  Pfliiger's  Arch.,  44;  Rohmann,  ibid.,  41. 

2  Rubner,  Zeitschr.  f.  Biologic,  15  and  19;  Constantinidi,  ibid.,  23;  Hultgren  and 
Lander gr en,  1.  c. 

^  Cavazzani,  Centralbl.  f.  Physiol.,  7.  See  foot-note  1,  p.  419;  also  Lombroso, 
Hofmeister's  Beitrage,  8. 

^  Munk,  Virchow's  Arch.,  SO.  See  also  v.  Walther,  Arch.,  f.  (Anat.  u.)  Physiol., 
1890;  Minkowski,  Arch.  f.  exp.  Path.  u.  Phann.,  21;  Frank,  Zeitschr.  f.  Biologic,  36; 
Moore,  see  Biochem.  Centralbl.,  1,  741-  Frank  and  Ritter,  Zeitschr.  f.  Biologic,  47. 


422  DIGESTION. 

The  assumption  that  the  fat  is  absorbed  chiefly  as  an  emulsion  is  partly 
based  on  the  abundance  of  emulsified  fat  in  the  chyle  after  feeding  with  fat, 
and  partly  on  the  fact  that  a  fat  emulsion  is  often  found  in  the  intestine 
after  such  food.  As  an  abundant  cleavage  of  neutral  fats  occurs  in  the 
intestinal  canal,  and  also  as  the  fatty  acids  do  not  occur  in  the  chyle  as 
such,  but  as  emulsified  fat  after  a  synthesis  with  glycerine  into  neutral  fats, 
it  is  to  be  doubted  whether  the  emulsified  fat  of  the  chyle  originates  from 
an  absorption  of  emulsified  fat  in  the  intestine  or  from  a  subsequent  emul- 
sification  of  neutral  fats  formed  synthetically.  This  doubt  has  greater 
warrant  in  that  Frank  i  has  shown  that  the  fatty-acid  ethyl  ester  is  abun- 
dantly taken  up  by  the  chyle  from  the  intestine,  not  as  such,  but  as  split-off 
fatty  acids  from  which  then  the  neutral  emulsified  fats  of  the  chyle  are 
formed. 

The  assumption  of  an  absorption  of  fats  as  an  emulsion  contradicts  the 
fact  that  an  emulsion  produced  by  means  of  soaps  is  not  permanent  in  an 
acid  liquid;  hence  we  cannot  consider  as  possible  the  presence  of  an  emul- 
sion in  the  intestine  so  long  as  it  is  acid.  This  difficulty  is  not  too  serious, 
as  the  reaction  is  often  due  to  only  carbonic  acid  and  bicarbonates  and 
also  as  found  by  KiJHXE  and  recently  shown  by  Moore  and  Krumbholz,^ 
the  proteins  have  a  preserving  action  upon  fat  emulsions.  The  older  views 
as  to  fat  absorption  were  that  the  fat  was  absorbed  as  soaps,  soluble  in 
water,  as  well  as  finely  emulsified  fat,  and  this  last  form  was  considered  as 
of  the  greatest  importance.  This  view  has  recently  undergone  essential 
modifications,  due  to  the  work  of  Moore  and  Rockwood,  and  especially 
to  the  extensive  work  of  Pfluger.^ 

Moore  and  Rockwood  have  shown  the  great  solvent  action  of  the  bile 
for  fatty  acids,  and  on  continuing  these  investigations  further,  Moore  and 
Parker  have  found  that  the  bile  increases  the  solubility  of  soaps  in  water 
and  can  prevent  their  gelatinization,  a  fact  which  is  of  greater  importance 
for  the  absorption  of  fats  than  the  solubility  of  the  fatty  acids  in  bile.  The 
quantity  of  lecithin  in  the  bile  is  of  great  importance  for  the  solubility  therein 
of  the  fatty  acids  as  well  as  the  soaps.  According  to  the  above-mentioned 
investigators,  the  absorption  of  fat  from  the  intestine  is  essentially  dependent 
upon  the  solubility  of  the  soaps  and  free  fatty  acids  in  the  bile.  The  neutral 
fats  are  split  and  the  free  fatty  acids  are  in  part  absorbed  dissolved  as  such 
by  the  bile,  and  in  part  combined  with  alkalies,  forming  soaps.  Neutral 
fats  are  regenerated  from  the  fatty  acids,  and  the  alkali  set  free  from  the 

*  Zeitschr.  f.  Biologie,  3(5. 

^Kiihne,  Lehrb.  der  physiol.  Chem.,  122;  Moore  and  Krumbholz,  Joum.  of  Phy- 
siol, 22. 

^  In  regard  to  the  newer  literature  on  fat  absorption,  see  the  works  of  Pfi  ger, 
Pfi  ger's  .^rch.,  SO,  §1,  82,  85,  88,  89,  and  90,  where  the  work  nf  other  investigators  is 
cited  and  discussed. 


ABSORPTION   OF  FATS.  423 

soaps  is  secreted  back  again  into  the  intestine  and  used  for  the  re-formation 
of  soaps. 

The  importance  of  the  bile,  the  soaps,  and  the  alkali  carbonates  has  been 
closely  studied,  chiefly  in  the  very  thorough  investigations  of  Pfluger. 
He  has  quantitatively  determined  the  solvent  power  of  the  above-men- 
tioned bodies — each  alone  as  well  as  different  mixtm-es  of  these — for  the 
various  fatty  acids,  and  has  closely  studied  the  mode  of  action  of  the  bile. 
From  his  investigations  he  has  arrived  at  the  conclusion  that  no  imsplit  fat 
is  absorbed,  that  all  fats,  before  their  absorption,  must  first  be  split  into 
glycerine  and  fatty  acids,  and  that  the  bile,  on  account  of  its  solvent 
power  for  soaps  and  fatty  acids,  is  sufficient  for  the  aljsorption  of  large 
quantities  of  fat  eaten.  The  object  of  the  formation  of  an  emulsion  is, 
according  to  this  view,  that  the  fat  in  this  condition  forms  such  a  large 
surface  for  the  action  of  the  steapsin  or  the  fat -splitting  agents. 

The  possibility  that  all  the  fat  must  be  first  split  and  that  no  unsplit 
fat  is  absorbed  is,  according  to  these  researches,  not  to  be  denied.  It  is 
the  opmion  of  the  author  that  it  is  still  too  early  to  give  a  positive  verdict 
as  to  how  these  conditions  in  the  intestine  are  brought  about  and  the  con- 
clusion must  be  left  for  fmther  investigations. 

The  next  question  is  whether  all  the  fat  or  the  greater  part  of  the  same 
passes  into  the  blood  throtigh  the  lymphatics  and  the  thoracic  duct. 
According  to  the  researches  of  Walther  and  Frank  i  on  dogs,  it  seems 
that  only  a  small  part  of  the  fats,  or  at  least  of  the  fatty  acids  fed  passes 
into  the  chylous  vessels;  but  these  obser^-ations  can  hardly  be  applied  to 
the  absorption  of  neutral  fats,  or  to  the  absorption  in  man  imder  normal 
circumstances.  Muxk  and  Rosexsteix,^  in  their  investigations  on  a 
girl  with  a  lymph  fistula  fotmd  60  per  cent  of  the  fat  ingested  in  the  chyle, 
and  of  the  total  quantity  of  fat  in  the  chyle  only  4-5  per  cent  existed  as 
soaps.  On  feeding  with  a  foreign  fatty  acid,such  as  erucic  acid,  they 
foimd  37  per  cent  of  the  introduced  body  as  neutral  fat  in  the  chvle. 

The  completeness  with  which  fats  are  absorbed  depends,  imder  normal 
conditions,  essentially  upon  the  kind  of  fat.  In  this  regard  it  is  known, 
especially  from  the  investigations  of  Muxk  and  Arxschixk,^  that  the 
varieties  of  fat  with  high  melting-points,  such  as  mutton-tallow  and  espe- 
cially stearin,  are  not  so  completely  absorbed  as  the  fats  with  low  melting 
points,  such  as  hog-  and  goose-fat,  olive-oil,  etc.  The  kind  of  fat  also  has 
an  influence  upon  the  rapidity  of  absorption,  as  ^Iuxk  and  Rosexstein 
found  that  solid  mutton-fat  was  absorbed  more  slowly  than  fluid  lipanin. 
The  extent  of  absorption  in  the  intestinal  tract  is,  under  physiological  con- 

1  Walther.  Arch.  f.  (Anat.  u.)  Physiol.,  1900;    Frank,  ibid.,  1892. 

2  Virchow's  Arch.,  123. 

'  Munk.  Virchow's  Arch..  80  and  95;  Arnschink.  Zeitschr.  f.  Biologie.  26. 


424  DIGESTION. 

ditions,  very  considerable.  In  the  case  of  a  dog  investigated  by  Voit  it 
was  found  that  out  of  350  grams  of  fat  (butter)  partaken,  346  grams  were 
absorbed  from  the  intestinal  canal,  and  according  to  the  investigations  of 
RuBNER  1  the  human  intestine  can  absorb  over  300  grams  of  fat  per  diem. 
The  fats  are,  according  to  Rubner,  much  more  completely  absorbed  when 
free,  in  the  form  of  butter  or  lard,  than  when  enclosed  in  cell-membranes, 
as  in  bacon. 

Claude  Bernard  showed  long  ago  with  exiDeriments  on  rabbits  in  which 
the  ductus  choledochus  was  made  to  open  into  the  small  intestine  above  the 
pancreatic  duct,  that  after  food  rich  in  fats  the  chylous  vessels  of  the  intes- 
tine above  the  pancreas  passages  were  transparent,  while  below  they  were 
milk-white,  and  also  that  the  bile  alone  cannot  produce  an  absorption  of 
the  emulsified  fat  without  the  pancreatic  juice.  Dastre  2  has  performed 
the  reverse  experiment  on  dogs.  He  tied  the  ductus  choledochus  and 
adjusted  a  biliary  fistula  so  that  the  bile  flowed  into  the  intestine  below 
the  mouth  of  the  pancreatic  passages.  On  killing  the  animal  after  a  meal 
rich  in  fat  the  chylous  vessels  were  first  found  milk-white  below  the  dis- 
charge of  the  biliary  fistula.  From  this  Dastre  draws  the  conclusion 
that  a  comliined  action  of  the  bile  and  pancreatic  juice  is  important  in  the 
absorption  of  fats — a  conclusion  which  stands  in  good  accord  with  the 
experience  of  many  others. 

Through  numerous  observations  of  many  investigators,  such  as  Bidder 
and  Schmidt,  Voit,  Rohmaxn,  Fr.  Muller,  I.  Muxk,^  and  others,  it  has 
been  shown  that  the  exclusion  of  the  bile  from  the  intestinal  tract  dimin- 
ishes the  absorption  of  fat  to  such  an  extent  that  only  one  seventh  to 
about  one  half  of  the  quantity  of  fat  ordinaril}^  absorbed  undergoes  al:)Sorp- 
tion.  In  icterus  with  entire  exclusion  of  the  bile,  a  considerable  decrease 
in  the  absorption  of  fat  is  noticed.  As  under  normal  conditions,  so  also  in 
the  absence  of  bile  in  the  intestine,  the  lower-melting  parts  of  the  fat  are 
more  completely  absorbed  than  those  which  have  a  high  melting-point. 
I.  MuNK  found  in  his  experiments  on  dogs  with  lard  and  mutton-tallow 
that  the  absorption  of  the  high-melting  tallow  was  reduced  twice  as  much 
as  the  lard  on  the  exclusion  of  the  bile  from  the  intestine. 

We  also  learn  from  the  investigations  of  Rohmann  and  I.  Munk  that 
in  the  absence  of  bile  the  relationship  between  fatty  acids  and  neutral  fats 
is  changed,  namely,  about  80-90  per  cent  of  the  fat  existing  in  the  faeces 
consists  of  fatty  acid,  while  under  normal  conditions  the  faeces  contain 
1  part  neutral  fat  to  about  2-2^  parts  free  fatty  acids.     It  is  not  possible 

*  Voit,  Zeitschr.  f.  Biologie,  9;   Rubner,  ibid.,  1.'). 
2  Arch,  (le  Physiol.  (5),  2. 

^F.  Muller,  Sitzungsber.  der  phys.-med.  Gesellscli.  zu  Wurzburg,  1885;  I.  Munk, 
Virchow's  Arch.,  122.     See  also  foot-notes  2  and  3,  p.  406. 


ABSORPTION  OF  FATS.  425 

to  state  how  this  increased  quantity  of  fatty  acids  in  the  fat  of  the  faeces 
is  produced  upon  the  exclusion  of  the  bile  from  the  intestine. 

There  is  no  doubt  that  the  bile  is  of  great  importance  in  the  absorption 
of  fats.  Still  there  is  also  no  doubt  that  rather  considerable  quantities  of 
fat  may  be  absorbed  from  the  intestine  in  the  absence  of  Ijile.  What  rela- 
tion does  the  pancreatic  juice  bear  to  this  fact? 

Upon  this  point  a  rather  large  number  of  observations  on  animals  have 
been  made  by  Abelmann  and  Minkowski,  Sandmeyer,  Harley,  Rosen- 
berg, Hedon  and  Ville,  and  also  on  man  by  Fr.  Muller  and  Deucher.i 
In  all  of  these  investigations  a  more  or  less  diminished  absorption  of  fat 
was  observed  after  the  extirpation  or  destruction  of  the  gland,  or  the 
exclusion  of  the  juice  from  the  intestine.  The  results  are  very  diverse 
as  to  the  extent  of  this  diminution,  as  in  certain  cases  no  absorption  of  fat 
was  observed,  while,  in  other  cases,  a  considerable  absorption  was  noted 
in  the  same  class  of  animal  (dog)  and  even  in  the  same  animal.  According 
to  Minkowski  and  Abelmann,  after  the  total  extirpation  of  the  pancreas 
the  fat  of  the  food  introduced  is  not  absorbed  at  all,  with  the  exception  of 
milk,  of  which  28-53  per  cent  of  the  fat  is  absorbed.  Other  investigators 
have  obtained  other  results,  and  Harley  has  observed  a  case  where  in  a 
dog  an  absorption  of  only  4  per  cent  of  the  milk-fat,  or,  on  the  complete 
exclusion  of  intestinal  bacteria,  even  no  absorption,  took  place.  The  con- 
ditions may  be  somewhat  different  in  the  different  cases;  but  it  is  certain 
that  the  absence  of  pancreatic  juice  from  the  intestine  essentially  affects 
the  fat  absorption.  It  is  also  just  as  certain  that  the  absorption  of  fat  is 
most  abundant  in  the  simultaneous  presence  of  bile  as  well  as  pancreatic 
juice  in  the  intestine.  A  little  fat  may  still  be  absorbed  even  in  the  absence 
of  these  two  fluids,  as  shown  by  the  investigations  of  HiiDON  and  Ville 

and  CUNNINGHAM.2 

The  reason  for  the  fact  that  the  fat  absorption  is  diminished  in  the 
absence  of  bile  from  the  intestine  must  be  sought  for  in  the  above-mentioned 
role  of  this  fluid.  It  is  more  difficult  to  state  why  the  absence  of  pan- 
creatic juice  causes  a  reduction  in  the  absorption  of  fat.  The  most  natural 
view  is  that  the  neutral  fats  are  here  less  completely  split,  but  this  does 
not  seem  to  be  the  case,  because  the  non-absorbed  fat  of  the  fseces  consists, 
on  the  exclusion  of  bile  and  pancreatic  juice  (Minkowski  and  Abelmann, 
Harley,  Hedon  and  Ville,  Deucher),  chiefly  of  free  fatty  acids.  A 
still  unknown  change  caused  by  gastric  lipase  or  by  micro-organisms  or 
otherwise  may  produce  a  cleavage  of  the  fat  in  these  cases.     The  imperfect 

^  Miiller,  "Unters.  iiber  den  Icterus,"  Zeitschr.  f.  klin.  Med.,  12;  Hedon  and  Ville, 
Arch,  de  Physiol.  (5),  9;  Harley,  Journ.  of  Physiol.,  18,  Joum.  of  Pathol,  and  Bacteriol., 
1895,  and  Proceed.  Roy.  Soc,  61.  In  regard  to  the  other  authors  see  foot-note  2, 
p.  418. 

^  Hedon  and  Ville,  1.  c. ;  Cunningham,  Journ.  of  Physiol.,  23. 


426  DIGESTION. 

fat  absorption  after  the  extirpation  of  the  pancreas  can  possibly  he  explained 
by  the  removal  of  a  considerable  part  of  the  alkalies  necessary  for  the 
formation  of  the  emulsion  and  for  the  solution  of  the  fatty  acids,  but  as 
Sandmeyer  found  in  dogs  deprived  of  their  pancreas  that  the  fat  absorption 
was  raised  by  giving  chopped  pancreas  with  the  fat,  this  can  hardly  be 
a  sufficient  explanation. 

The  soluble  salts  are  also  absorbed  with  the  water.  The  proteins, 
which  can  dissolve  a  considerable  quantity  of  salts,  such  as  earthy  phos- 
phates which  are  otherwise  insoluble  in  alkaline  water,  are  of  great  impor- 
tance in  the  absorption  of  such  salts. 

The  soluble  constituents  of  the  digestive  secretions  may,  like  other 
dissolved  bodies,  be  absorbed,  as  is  demonstrated  by  the  passage  of  pepsin 
into  urine;  the  enzymes  may  also  be  absorbed.  The  occurrence  of  uro- 
bilin in  urine  attests  the  absorption  of  the  bile-constituents  under  physio- 
logical conditions  despite  the  fact  that  the  occurrence  of  very  small  traces 
of  bile-acids  in  the  urine  is  disputed.  The  absorption  of  bile-acids  by  the 
intestine  seems  to  be  positively  proved  by  other  observations.  Tap- 
PEiNER  1  introduced  a  solution  of  bile-salts  of  a  known  concentration  into 
an  intestinal  knot  and  after  a  time  investigated  the  contents.  He  found 
that  in  the  jejunum  and  the  ileum,  but  not  in  the  duodenum,  an  absorption 
of  bile-acids  took  place,  and  further  that  of  the  two  bile-acids  only  the 
glycocholic  acid  was  absorbed  in  the  jejunum.  Further,  Schiff  long  ago 
expressed  the  opinion  that  bile  undergoes  an  intermediate  circulation,  in 
such  wise  that  it  is  absorbed  from  the  intestine,  then  carried  to  the  liver 
by  the  blood,  and  lastly  eliminated  from  the  blood  by  this  organ.  Although 
this  view  has  met  with  some  opposition,  still  its  correctness  seems  to  be 
established  by  the  researches  of  various  investigators,  and  more  recently 
by  Prevost  and  Binet,  and  specially  by  Stadelmann  and  his  pupils.^ 
After  the  introduction  of  foreign  bile  into  the  intestine  of  an  animal  the 
foreign  bile-acids  appear  again  in  the  secreted  bile. 

How  does  the  removal  of  large  portions  of  the  various  parts  of  the 
intestine  affect  absorption?  Harley^  has  been  able  to  perform  a  partial 
extirpation  of  the  large  intestine  and  in  another  instance  a  complete  extir- 
pation. This  last  condition  increased  the  faeces  considerably,  especially 
because  of  the  large  increase  in  the  water  (fivefold).  Fats  and  carbohy- 
drates were  absorbed  just  as  completely  as  in  the  normal.  The  absorp- 
tion of  the  proteins,  on  the  contrary,  was  reduced  to  only  84  per  cent  as 
compared  to  93-98  per  cent  in  normal  dogs.     After  extirpation  the  fseces 


'  Wien.  Sitzungsber.,    77. 

^  Schiff,  Pfliger's  Arch.,  3;    Prevost  and   Binet,  Compt     rend.,  106;    Stadelmann, 
see  foot-note  1 ,  p.  309. 

^  Proceed.  Roy.  Soc,  64. 


ABSORPTION    IX   DIFFERENT   PARTS   OF  THE   INTESTINE.   427 

sometimes  did  not  contain  any  m-obilin  or  only  traces  thereof,  while  bile- 
pigments  existed  in  large  amounts. 

Erlanger  and  Hewlett  ^  found  that  dogs,  from  which  70-83  per  cent 
of  the  total  length  of  the  jejunum  and  ileum  had  been  removed,  could  be 
kept  alive  like  other  animals  if  only  the  food  was  not  too  rich  in  fat.  When 
the  food  contained  large  amounts  of  fat  then  25  per  cent  was  evacuated 
by  the  fseces  as  compared  to  4-5  per  cent  in  the  normal  animal.  Under 
these  same  conditions  the  amount  of  nitrogen  in  the  faeces  was  increased 
to  twice  the  normal    amount. 

After  the  exclusion  of  the  colon  in  rabbits,  Bergmann  and  Hultgren  ^ 
could  find  no  definite  action  upon  the  availability  of  the  cellulose  and 
also  no  diminution  in  the  utility  of  the  other  constituents  of  the  food  could 
be  observ-ed.  Zuntz  and  Ustjanzew  3  also  found  that  the  removal  of  the 
caecum  had  no  influence  on  the  utilization  of  nitrogen;  but  in  regard  to 
other  points  they  arrived  at  different  results.  They  fomid,  namely,  that 
the  caecum  of  the  rodent  is  of  great  importance  for  the  digestion  of  crude 
fibre  and  the  pentosanes.  On  feeding  hay  and  wheat  to  rabbits  after  the 
removal  of  the  caecum,  the  digestion  coefficient  for  crude  fibre  fell  from 
42.8  to  23.4-18.7  per  cent  and  for  pentosanes  from  50  to  40-28.7  per  cent. 

The  question  as  to  the  forces  which  are  active  in  the  intestine  during 
absorption  has  not  been  answered.  It  is  certain  that  thus  far  the  laws  of 
diffusion  and  osmosis  alone  are  not  sufficient  to  explain  absorption,  although 
the  views  are  disputed.  With  all  these  facts  in  view,  and  as  it  is  not  within 
the  scope  of  this  book  to  enter  more  in  detail  upon  the  numerous  inves- 
tigations on  this  subject,  we  must  refer  to  larger  works  ^  and  to  text-books 
on  physiology  for  further  information. 

*  Amer.  Journ.  of  Physiol.,  6. 
^Skand.  Arch.  f.  Physiol,  U. 

^  Verhandl.  d.  physiol.  Gesellsch.  zu  BerHn,  1904-1905. 

*  See  Hober,  Physikalische  Cliemie  der  Zelle,  Leipzig,  1906,  and  I.  Munk,  Ergeb- 
nisse  der  Physiologie,  I,  Abt.  1;  Hamburger,  Osmotischer  Druck  und  lonenlehre, 
Bd.  2,  Wiesbaden,  1904. 


CHAPTER  X. 

TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

I.    The  Connective  Tissues. 

The  form-elements  of  the  typical  connective  tissues  are  cells  of  various 
kinds,  of  a  not  very  well-known  chemical  composition,  and  gelatine-yielding 
fibrils,  which,  like  the  cells,  are  imbedded  in  an  interstitial  or  intercellular 
substance.  The  fibrils  consist  of  collagen.  The  interstitial  substance  con- 
tains chiefly  mucoid  {tendon-mucoid) ,  besides  serglobidin  and  seralbumin, 
which  occur  in  the  parenchymatous  fluid  (Loebisch  ^). 

The  connective  tissue  also  often  contains  fibres  or  formations  consisting 
of  elastin,  sometimes  in  such  great  quantities  that  the  connective  tissue 
is  transformed  into  elastic  tissue.  A  third  variet}^  of  fibres,  the  reticular 
fibres,  also  occurs,  and  according  to  Siegfried  these  consist  of  reticulin. 

If  finely  divided  tendons  are  extracted  in  cold  water  or  NaCl  solutions, 
the  protein  bodies  soluble  in  the  nutritive  fluid  in  addition  to  a  little  mucoid 
are  dissolved.  If  the  residue  is  extracted  with  half-saturated  lime-water, 
then  the  mucoid  is  dissolved  and  may  be  precipitated  from  the  filtered 
extract  by  adding  an  excess  of  acetic  acid.  The  extracted  residue  con- 
tains the  fibrils  of  the  connective  tissue  together  with  the  cells  and  the 
elastic  substance. 

The  so-called  tendon  mucin  is  not  true  mucin,  but  a  mucoid,  which, 
as  first  shown  b}'  Levexe  and  then  by  Cutter  and  Gies,  contains  a  part  of 
its  sulphur  as  an  acid  related  to  chondroitin-sulphuric  acid.  These  mucoids, 
which  according  to  Cutter  and  Gies  are  mixtures  of  several  glucoproteids, 
contain  2.2-2.33  per  cent  sulphur,  as  shown  by  the  analyses  of  Chitten- 
den and  Gies,  as  well  as  those  of  Cutter  and  Gies.  The  quantity  of 
sulphur  split  off  as  sulphuric  acid  was  1.33-1.62  per  cent  (Cutter  and 
Gies  2). 

The  fibrils  of  the  connective  tissue  are  elastic  and  swell  slightly  in  water, 
somewhat  more  in  dilute  alkalies  or  in  acetic  acid.  On  the  other  hand, 
they  shrink  by  the  action  of  certain  metallic  salts,  such  as  ferrous  sulphate 

*  Zeitschr.  f.  physiol.  Chem.,  10. 

^  Levene,  ibid.,  31  and  39;  Cutter  and  Gies,  Amer.  Joum.  of  Physiol.,  6;  Chittenden 
and  Gies,  Joum.  of  exp.  Med.,  1. 

428 


CONNECTIVE  TISSUES.  429 

or  mercuric  chloride,  and  tannic  acid,  which  form  insoluble  compounds 
with  the  collagen.  Among  these  compounds,  which  prevent  putrefaction 
of  the  collagen,  that  with  tannic  acid  has  been  fomid  of  the  greatest  tech- 
nical importance  in  the  preparation  of  leather.  In  regard  to  the  collagens, 
gelatines,  elastins,  and  reticulins,  see  pages  75  to  81. 

The  tissues  described  under  the  names  mucous  or  gelatinous  tissues  are 
characterized  more  by  their  physical  than  by  their  chemical  properties  and 
have  been  but  little  studied.  This  much,  however,  is  known,  that  the 
mucous  or  gelatinous  tissues  contain,  at  least  in  certain  cases,  as  in  the 
acalephae,  no  mucin. 

The  lunbilical  cord  is  the  most  accessible  material  for  the  investigation 
of  the  chemical  constituents  of  the  gelatinous  tissues.  The  mucin  occiuring 
therein  has  been  described  on  page  C7.  C.  Th.  Morxer  ^  has  found  a 
mucoid  in  the  vitreous  humor  which  contains  12.27  i>er  cent  nitrogen  and 
1.19  per  cent  sulphur. 

Yomig  connective  tissue  is  richer  in  mucoid  than  old.  Halliburton  ^ 
found  an  average  of  7.66  p.  m.  mucoid  in  the  skin  of  verj^  3'oung  children 
and  only  3.85  p.  m.  in  the  skin  of  adults.  In  so-called  myxoedema,  in 
which  a  re-formation  of  the  connective  tissue  of  the  skin  takes  place,  the 
ciuantity  of  mucoid  is  also  increased. 

The  connective  tissue  and  also  the  elastic  tissue  are  richer  in  water  and 
poorer  in  sohds  in  yoimg  animals  as  compared  with  f ull-growTi  animals.  This 
may  be  seen  from  the  following  analyses  of  the  Achilles  tendon  (Buerger 
and  GiEs)  and  of  the  ligamentum  nuchse  (Vaxdegrift  and  Gies  ^). 


Achill 

es  tendon. 

Calf. 

Ox. 

Water 

.   675.1  p.  m. 

628.7  p.m. 

Solids 

.    324.9    " 

371.3     " 

Organic  bodies .  .  .  . 

.   318.4    " 

366.6     " 

Inorganic  bodies  .  . 

.       6.1    " 

4.7     " 

Fat 

10.4     " 

Proteid 

9    9      " 

ilucoid 

12.83  " 

Elastin 

16.33  " 

Collagen 

315  88  " 

Extractives,  etc  .  .  . 

8.96  " 

Ligament. 

Calf. 

Ox. 

651.0  p.  m. 

575.7  p.  m 

394.0    " 

424.3     " 

342.4    " 

419.6     " 

6.6    " 

4.7     " 

(( 

11.2     " 

6.16  " 

5.25  " 

316.70  " 

72.30  " 

7.99  " 

In  regard  to  the  mineral  bodies  it  must  be  remarked  that  according  to 
the  determinations  of  H.  Schulz^  the  connective  tissue  is  rich  in  silicic 
acid.  The  greatest  amount  was  fotmd  by  him  in  the  crj-stalline  lens  of 
the  ox,  namely,  0.5814  gram  per  kilo  of  dried  substance.     In  man  he  found 

*  Zeitschr.  f.  physiol.  Cliem.,  18,  250. 

'  Mucin  in  Myxoedema :  Further  Analyses.  King's  College  Collected  Papers  No.  1, 
1893. 

^  Buerger  and  Gies,  Amer.  Joum.  of  Piiysiol..  6;  Vandegrift  and  Gies,  ibid.,  5. 

*  PflLiger's  .\rch.,  S4  and  S9. 


430  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

0.0637  gram  in  the  tendons,  0.1064  gram  in  the  fascia,  and  0.244  gram  in 
Wharton's  jelly  for  every  kilo  of  dried  substance.  The  quantity  of  silicic 
acid  is  higher  in  the  young  than  in  the  old;  in  man  it  is  highest  in  the 
embryonic  connective  tissue  of  the  umbilical  cord.  In  the  last-named 
substance  Schulz  found  also  0.403  gram  FeoOa,  0.693  gram  MgO,  3.297 
grams  CaO,  and  3.794  grams  P2O5  for  every  kilo  of  dried  substance, 

n.    Cartilage. 

Cartilaginous  tissue  consists  of  cells  and  an  original  hyaline  matrix, 
which,  however,  may  become  changed  in  such  wise  that  there  appears  in  it 
a  network  of  elastic  fibres  or  connective-tissue  fibrils. 

Those  cells  that  offer  great  resistance  to  the  action  of  alkalies  and 
acids  have  not  been  carefully  studied.  According  to  former  \'iew^s,  the 
matrix  was  considered  as  consisting  of  a  body  analogous  to  collagen, 
so-called  chondrigen.  The  recent  investigations  of  Morochowetz  and 
others,  but  especially  those  of  C.  Morner,i  have  shown  that  the  matrix  of 
the  cartilage  consists  of  a  mixture  of  collagen  with  other  bodies. 

The  tracheal,  thyroideal,  cricoidal,  and  ar5i:enoidal  cartilages  of  full- 
grown  cattle  contain,  according  to  Morner,  four  constituents  in  the  matrix 
namely,  chondromucoid,  chondroitin-sulphuric  acid,  collagen,  and  an  albu- 
minoid. 

Chondromucoid.  This  body,  according  to  Morner,  has  the  composi- 
tion C  47.30,  H  6.42,  N  12.58,  S  2.42,  O  31.28  per  cent.  Sulphur  is  in  part 
loosely  combined  and  may  be  split  off  by  the  action  of  alkalies,  and  a  part 
separates  as  sulphuric  acid  w^hen  boiled  with  hydrochloric  acid.  Chondro- 
mucoid is  decomposed  by  dilute  alkalies  and  yields  alkali  albuminate, 
peptone  substances,  chondroitin-sulphuric  acid,  alkali  sulphides,  and 
some  alkali  sulphates.  On  boiling  with  acids  it  yields  acid  albuminate, 
peptone  substances,  chondroitin-sulphuric  acid,  and  on  account  of  the 
further  decomposition  of  this  last  body,  sulphuric  acid  and  a  reducing 
substance  are  formed. 

Chondromucoid  is  a  white,  amorphous,  acid-reacting  powder  which  is 
insoluble  in  water,  but  dissolves  easily  on  the  addition  of  a  little  alkali. 
This  solution  is  precipitated  bj^  acetic  acid  in  great  excess  and  by  small 
quantities^  of  mineral  acids.  The  precipitation  may  be  retarded  by  neutral 
salts  or  b,v  chondroitin-sulphuric  acid.  The  solution  containing  NaCl  and 
acidified  with  HCl  is  not  precipitated  by  potassium  ferrocyanide.  Precipi- 
tants  for  chondromucoid  are  alum,  ferric  chloride,  sugar  of  lead,  or  basic 
lead  acetate.     Chondromucoid  is  not  precipitated  by  tannic  acid,  and  it 

*  Morochowetz,  Verhandl.  d.  naturh.  med.  Vereins  zu  Heidelberg,  1,  Heft  5;  Morner, 
Skand.  Arch.  f.  Physiol.,  1. 


CHONDROITIN-SULPHURIC  ACID.  431 

may  by  its  presence  prevent  the  precipitation  of  gelatine  by  this  acid.  It 
gives  the  usual  color  reactions  for  proteins,  namely,  with  nitric  acid,  with 
copper  sulphate  and  alkali,  with  Millon's  and  Adamkiewicz's  reagents. 

Chondroitin-sulphuric  Acid,  chondroitic  acid.  This  acid,  which  was 
first  prepared  pure  from  cartilage  by  C.  Morxer  and  identified  by  him  as 
an  ethereal  sulphuric  acid,  occurs,  according  to  Morner,  in  all  varie- 
ties of  cartilage  and  also  in  the  tunica  intima  of  the  aorta  and  as  traces 
in  the  bone  substance.  K.  Morxer  has  also  found  it  in  the  ox-kidney 
and  in  human  urine  as  a  regular  constituent.  According  to  Krawkow, 
who  found  it  in  the  cervical  ligament  of  the  ox,  it  combines  with  proteid, 
forming  amyloid  (see  page  69),  which  explains  the  occurrence  of  this  body 
in  amyloid-degenerated  livers,  as  observed  by  Oddi.^  The  identity  of  the 
ethereal  sulphuric  acid  occurring  in  liver  amyloid  with  chondroitin-sul- 
phuric acid  does  not  seem  to  be  quite  clear,  according  to  the  researches  of 
MoNERY.  According  to  Levexe,^  the  glucothionic  acid  which  is  prepared 
from  tendon  mucoid  and  which  gives  the  orcin  reaction  for  glucuronic  acid, 
and  yields  furfurol  on  distillation  with  hydrochloric  acid,  is  not  identical 
with  the  chondroitin-sulphuric  acid,  but  is  probably  related  thereto. 

Chondroitin-sulphuric  acid  has  the  formula  C18H27NSO17,  according  to 
Schmiedeberg.3  As  primary  products  this  acid  yields  ©n  cleavage  sulphuric 
acid  and  a  nitrogenous  substance,  chondroitin,  according  to  the  following 
equation : 

CisHoyNSOiT  +H20  =  H2S04  +  Ci8H27NOi4. 

Chondroitin,  which  is  similar  to  gum  arable  and  which  is  a  monobasic  acid, 
yields  acetic  acid  and  a  new  nitrogenous  substance,  chondrosin,  as  cleavage 
products,  on  decomposition  with  dilute  mineral  acids: 

Ci8H27NOi4  +  3H20  =  3C2H402  +  Ci2H2iNOii. 

Chondrosin,  which  is  also  a  gummy  substance  soluble  in  water,  is  a  mono- 
basic acid  and  reduces  copper  oxide  in  alkaline  solution  even  more  strongly 
than  dextrose.  It  is  dextrogyrate  and  represents  the  reducing  substance 
obtained  by  previous  investigators  in  an  impm'e  form  on  boiling  cartilage 
with  an  acid.  The  products  obtained  on  decomposing  chondrosin  with 
barium  hydrate  tend  to  show,  according  to  Schmiedeberg,  that  chondro- 
sin contains  the  atomic  groups  of  glucuronic  acid  and  glucosamine.  This 
assumption  does  not  seem  to  have  sufficient  foundation.  According  to 
Ogler  and  Neuberg,'*  chondrosin  does  not  give  the  orcin  test  nor  does 

»C.  Morner,  1.  c,  and   Zeitschr.  f.  physiol.  Chem.,  20  and   23:    K.  Morner,  Skand. 
Arch.  f.  Physiol.,  6;  Krawkow,  Arch.  f.  exp.  Path.  u.  Pharm.,  40;  Oddi,  ihid.,  33. 
2  Mon^ry,  Compt.  rend.  soc.  biol.,  .54;   Levene,  Zeitschr.  f.  physiol.  Chem.,  39. 
^  Arch.  f.  exp.  Path.  u.  Pharm.,  28. 
*  Zeitschr.  f.  physiol.  Chem.,  37. 


432  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

it  yield  furfurol.  It  contains  neither  glucuronic  acid  nor  glucosamine,  and 
on  cleavage  with  baryta  it  yields,  besides  a  carbohydrate  complex  which 
has  not  been  studied,  an  oxyamino-acid  having  the  formula  CeHisOeN; 
also  a  hexosamine  acid  or  tetraoxyaminocaproic  acid. 

Chondroitin-sulphuric  acid  appears  as  a  white  amorphous  powder, 
which  dissolves  very  easily  in  water,  forming  an  acid  solution  and,  when 
sufficiently  concentrated,  a  sticky  liquid  similar  to  a  solution  of  gum  arable. 
Nearly  all  of  its  salts  are  soluble  in  water.  The  neutralized  solution  is 
precipitated  by  tin  chloride,  basic  lead  acetate,  neutral  ferric  chloride, 
and  by  alcohol  in  the  presence  of  a  little  neutral  salt.  The  solution,  on 
the  other  hand,  is  not  precipitated  by  acetic  acid,  tannic  acid,  potassium 
ferrocyanide  and  acid,  sugar  of  lead,  mercuric  chloride,  or  silver  nitrate. 
Acidified  solutions  of  alkali  chondroitin-sulphates  cause  a  precipitation 
when  added  to  solutions  of  gelatine  or  proteid. 

Chondromucoid  and  chondroitin-sulphuric  acid  may  be  prepared,  accord- 
ing to  MoRNER,  by  extracting  finely  cut  cartilage  with  water,  which  dis- 
solves the  preformed  chondroitin-sulphuric  acid  besides  some  chondro- 
mucoid. In  this  watery  extract,  the  chondroitin-suliDhuric  acid  prevents 
the  precipitation  of  the  chondromucoid  by  means  of  an  acid.  If  2-4  p.  m. 
HCl  is  added  to  this  watery  extract  and  warmed  on  the  water-bath,  the 
chondromucoid  gradually  separates,  while  the  chondroitin-sulphuric  acid 
and  the  rest  of  the  chondromucoid  remain  in  the  filtrate.  If  the  cartilage, 
which  has  been  lixiviated  with  water,  at  the  temperature  of  the  body,  is 
extracted  with  hydrochloric  acid  of  2-3  p.  m.  until  the  collagen  is  con- 
verted into  gelatine  and  dissolved,  the  remaining  chondromucoid  may  be 
removed  from  the  insoluble  residue  by  dilute  alkali  and  precipitated  from 
the  alkaline  extract  by  an  acid.  It  may  be  purified  by  repeated  solution 
in  water  with  the  aid  of  a  little  alkali,  and  precipitation  with  an  acid,  and 
then  finally  by  extraction  with  alcohol  and  ether. 

The  pre-existing  chondroitin-sulphuric  acid,  or  that  formed  by  the 
decomposition  of  chondromucoid,  is  obtained  by  lixiviating  the  cartilage 
with  a  5  per  cent  caustic-alkali  solution.  The  alkali  albuminate  formed 
by  the  decomposition  of  the  chondromucoid  can  be  removed  from  the 
solution  by  neutralization,  then  the  peptone  precipitated  by  tannic  acid, 
the  excess  of  this  acid  removed  with  sugar  of  lead,  and  the  lead  separated 
from  the  filtrate  by  H2S.  If  further  purification  is  necessar}^  the  acid  is 
precipitated  with  alcohol,  the  precipitate  dissolved  in  water,  this  solution 
dialyzed  and  precipitated  again  with  alcohol, — this  solution  in  water  and 
precipitation  with  alcohol  being  repeated  a  few  times, — and  lastly  the  acid 
is  treated  with  alcohol  and  ether. 

ScHMiEDEBERG  prepared  the  acid  from  the  septum  narium  of  the  pig 
according  to  the  following  method:  The  finely  divided  cartilage  is  first 
exposed  to  artificial  peptic  digestion,  then  carefully  washed  with  water 
and  the  insoluble  residue  treated  with  2-3  per  cent  hydrochloric  acid. 
This  cloudy  liquid  containing  hydrochloric  acid  is  precipitated  with  alcohol 
(about  ^  vol.)  and  the  clear  filtrate  treated  with  absolute  alcohol  and 
some  ether.    The   precipitate,  consisting  chiefly  of  a  combination  or  a 


COLLAGEN  AND  ALBUMINOID  OF  CARTILAGE.  433 

mixture  of  chondroitin-sulphuric  acid  and  gelatine  peptone  (peptochondrin), 
is  first  washed  with  alcohol  and  then  with  water.  It  is  then  dissolved  in 
alkaline  water  and  the  basic  alkali  compound  precipitated  from  this 
solution  by  the  addition  of  alcohol,  whereby  the  gelatine-peptone  alkali 
remains  in  solution.  The  precipitate  is  purified  by  repeated  solution  in 
alkaline  water  and  precipitated  by  alcohol.  To  obtain  chondroitin-sul- 
phuric acid  entirely  free  from  chondroitin  it  is  more  advantageous  to 
prepare  the  potassium-copper  compound  of  the  acid  from  the  alkaline 
solution  by  the  alternate  addition  of  copper  acetate  and  caustic  potash 
and  precipitation  with  alcohol.  The  reader  is  referred  to  the  original 
article  for  more  details  and  also  for  Oddi's  method. 

The  collagen  of  the  cartilage  gives,  according  to  Morner,  a  gelatine  which 
contains  only  16.4  per  cent  N  and  which  can  hardly  be  considered  identical 
with  ordinary  gelatine. 

In  the  above-mentioned  cartilages  of  full-grown  animals  the  chondroitin- 
sulphuric  acid  and  chondromucoid,  perhaps  also  the  collagen,  are  found 
surrounding  the  cells  as  romid  balls  or  lumps.  These  balls  (Morner's 
chondrin-halls) ,  which  give  a  blue  color  with  methyl-violet,  lie  in  the  meshes 
of  a  trabecular  structure,  which  is  colored  when  brought  in  contact  with 
tropaeolin. 

The  albuminoid  is  a  nitrogenized  body  which  contains  loosely  com- 
bined sulphur.  It  is  soluble  with  difficulty  in  acids  and  alkalies  and 
resembles  keratin  in  many  respects,  but  differs  from  it  by  being  soluble 
in  gastric  juice.  In  other  respects  it  is  more  similar  to  elastin,  but  differs 
from  this  substance  by  containing  sulphur.  This  albuminoid  gives  the 
color  reactions  of  the  protein  bodies. 

The  preparation  of  cartilage  gelatine  and  the  albuminoid  may  be  per- 
formed according  to  the  following  method  of  Morner:  First  remove  the 
chondromucoid  and  chondroitin-sulphuric  acid  by  extraction  with  dilute 
caustic  potash  (0.2-0.5  per  cent),  remove  the  alkali  from  the  remaining 
cartilage  by  water,  and  then  boil  with  water  in  a  Papin's  digester.  The 
collagen  passes  into  solution  as  gelatine,  while  the  albuminoid  remains 
undissolved  (contaminated  by  the  cartilage-cells).  The  gelatine  may  be 
purified  by  precipitating  with  sodium  sulphate,  which  must  be  added  to 
saturation  in  the  faintly  acidified  solution,  redissolving  the  precipitate  in 
water,  dialyzing  well,  and  precipitating  with  alcohol. 

According  to  Morner,  no  albuminoid  is  found  in  young  cartilage,  but 
only  the  three  first -mentioned  constituents.  Nevertheless  the  young  carti- 
lage contains  about  the  same  amounts  of  nitrogen  and  mineral  substances 
as  the  old.  The  cartilage  of  the  ray  {Raja  hatis  Lin.),  which  has  been 
investigated  by  Lonnberg,^  contains  no  albuminoid  and  only  a  little 
chondromucoid,  but  a  large  proportion  of  chondroitin-sulphuric  acid  and 
collagen. 

•  Maly's  Jahresber.,  19,  325. 


434  TISSUES  OF   THE  CONNECTIVE   SUBSTANCE. 

According  to  Pfluger  and  Handel,'  glycogen  occurs  to  a  slight  extent 
in  all  matrices,  and  of  these  it  is  richest  in  the  cartilage.  Tendons,  liga- 
ment um  nuchse,  and  cartilage  of  the  ox  contained  0.06,  0.07,  and  2.17  p.  m. 
glycogen  respectively  (Handel). 

Hoppe-Seyler  found  in  fresh  human  rib-cartilage  676.7  p.  m.  water, 
301.3  p.  m.  organic  and  22  p.  m.  inorganic  substance,  and  in  the  cartilage 
of  the  knee-joint  735.9  p.  m.  water,  248.7  p.  m.  organic  and  15.4  p.  m. 
inorganic  substance.  Pickardt  found  402-574  p.  m.  water  and  72.86  p.  m. 
ash  (no  iron)  in  the  laryngeal  cartilage  of  oxen.  The  ash  of  cartilage  con- 
tains considerable  amounts  (even  800  p.  m.)  of  alkali  sulphate,  which 
probably  does  not  exist  originally  as  such,  but  is  produced  in  great  part  by 
the  incineration  of  the  chondroitin-sulphuric  acid  and  the  chondromucoid. 
The  analyses  of  the  ash  of  cartilage  therefore  cannot  give  a  correct  idea  of 
the  quantity  of  mineral  bodies  existing  in  this  substance.  The  cartilage 
is  richest  in  sodium  of  all  the  tissues  of  the  body,  and  according  to  Bunge  ^ 
the  amount  of  Na  and  CI  is  greatest  in  young  animals.  In  1000  parts  of 
cartilage  dried  at  120°  C,  Bunge  found  91.26  parts  NagO  in  the  shark,  33.98 
in  the  ox  embryo,  32.45  in  a  fourteen-day-old  calf,  and  26.4  in  a  ten-weeks- 
old  calf. 

The  Cornea.  The  corneal  tissue,  which  is  considered  by  many  investi- 
gators to  be  related  to  cartilage  in  a  chemical  sense,  contains  traces  of 
proteid  and  a  collagen  as  chief  constituent,  which  C.  Morner^  claims 
contains  16.95  per  cent  N.  According  to  him  it  also  contains  a  mucoid 
which  has  the  composition  C  50.16,  H  6.97,  N  12.79,  and  S  2.07  per  cent. 
On  boiling  with  dilute  mineral  acid  this  mucoid  yields  a  reducing  sub- 
stance. The  globulins  found  by  other  investigators  in  the  cornea  are  not 
derived  from  the  matrix,  according  to  Morner,  but  from  the  layer  of 
epithelium.  According  to  Morner,  Descemet's  membrane  consists  of 
membranin  (page  69),  which  contains  14.77  per  cent  N  and  0.90  per  cent  S. 

In  the  cornea  of  oxen  His  *  found  758.3  p.  m.  water,  203.8  p.  m.  gelatine- 
forming  substance,  28.4  p.  m.  other  organic  substance,  besides  8.1  p.  m. 
soluble  and  1.1  p.  m.  insoluble  salts. 

III.    Bone. 

The  bony  structure  proper,  when  free  from  other  formations  occurring 
in  bones,  such  as  marrow,  nerves,  and  blood-vessels,  consists  of  cells  and  a 
matrix. 


'  Pfl  :ger's  Arch.,  i)2;   Handel,  ibid. 

2  Hoppe-Seyler,  cited  from  Kiihne's  Lehrbuch  d.  physiol.  Chem.,  387;    Pickardt, 
Centralbl.  f.  Physiol.,  Ci,  735;  Bunge,  Zeitschr.  f.  physiol.  Chem.,  28. 

3  Zeitschr.  f.  physiol.  Chem.,  IS. 

■•Cited  from  Gamgee,  Physiol.  Chem.,  1880,  451. 


BONE.  435 

The  cells  have  not  been  closely  studied  in  regard  to  their  chemical  con- 
stitution. On  boiling  with  water  they  yield  no  gelatine.  They  contain  no 
keratin,  which  is  not  usually  present  in  the  bony  structure  (Herbert 
Saoth  ^). 

The  ruatriz  of  the  bony  structure  contains  two  chief  constituents, 
namely,  an  organic  substance,  and  the  so-called  bone-earths,  lime-salts, 
enclosed  in  or  combined  with  it.  K  bones  are  treated  with  dilute  hydro- 
chloric acid  at  the  ordinary  temperature,  the  lime-salts  are  dissolved  and 
the  organic  substance  remains  as  an  elastic  mass,  preserving  the  shape 
of  the  bone. 

The  organic  matrix  consists  chiefly  of  ossein,  which  is  generally  con- 
sidered as  identical  with  the  collagen  of  the  connective  tissue.  It  also 
contains,  as  Hawk  and  Gies  ^  have  shown,  mucoid  and  albuminoid.  After 
the  removal  of  the  lime-salts  by  hydrochloric  acid  of  2-5  p.  m.  these  experi- 
menters were  able  to  extract  the  mucoid  by  one-half  saturated  lime-water 
and  to  precipitate  it  with  2  p.  m.  hydrochloric  acid.  After  the  removal 
of  the  osseomucoid  and  collagen  (by  boiling  with  water)  they  ol3tained 
the  albuminoid  as  an  insoluble- residue. 

The  osseomucoid  on  boiling  with  hydrochloric  acid  yielded  a  reducing 
substance  and  sulphuric  acid,  1.11  per  cent  sulphur  appearing  in  this 
form.  The  osseomucoid  stands  close  to  the  chondro-  and  tendon  mucoid 
in  elementar}'  composition,  as  may  be  seen  from  the  following  analyses: 

o 

31.40  (Hawk  and  Gies) 
31.28  (C.  Morner) 
30.60  (Chittexdex  and  Gies) 
28.01  (C.  Morner) 

The  osseoalbuminoid  is  insoluble  in  2  p.  m.  hydrochloric  acid  and  in  5  p.  m. 
Na2C03,  but  dissolves  in  10  per  cent  KOH  with  the  formation  of  albumin- 
ates.    The  composition  of  chondro-  and  osseoalbuminoid  is  as  follows: 

c 

Osseoalbuminoid 50. 16 

Chondroalbuminoid 50 .  46 

The  inorganic  constituents  of  the  bony  structure,  the  so-called  bone- 
earths,  which  after  the  complete  calcination  of  the  organic  substance 
remain  as  a  white,  brittle  mass,  consist  chiefly  of  calcium  and  phosphoric 
acid,  but  also  contain  carbonic  acid  and,  in  smaller  amounts,  magnesium. 
chlorine,  and  fluorine.  Alkali  sulphate  and  iron,  which  have  been  found 
in  bone-ash,  do  not  seem  to  belong  exactly  to  the  bony  substance,  but  to 
the  nutritive  fluids  or  to  the  other  constituents  of  bones.     The  traces  of 

'  Zeitschr.  f.  Biologic,  19.  '  Amer.  Joum.  of  Physiol.,  5  and  7. 


C 

H 

N 

S 

Osseomucoid 

.   47.43 

6.63 

12.22 

2.32 

Chondromucoid.  . 

.    47.30 

6.42 

12.58 

2.42 

Tendon  mucoid.  . 

.     48.76 

6.53 

11.75 

2.33 

Corneal  mucoid.  . 

50.16 

6.97 

12.79 

2.07 

H 

N 

S 

0 

7.03 

16.17 

1.18 

25 .  46  \  Hawk  and 

7.05 

14.95 

1.86 

25 .  68  /       Gies 

436  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

sulphate  occurring  in  the  boue-ash  are  derived,  according  to  Morner,i 
from  the  chondroitin-sulphuric  acid.  According  to  Gabriel,  potassium 
and  sodium  are  essential  constituents  of  bone-earth,  and  this  has  been 
substantiated  by  Aron.^ 

The  opinions  of  investigators  differ  somewhat  as  to  the  manner  in  which 
the  mineral  bodies  of  the  bony  structure  are  combined  with  each  other. 
Chlorine  is  present  in  the  same  form  as  in  apatite  (CaCl2,3Ca3P208).  If 
we  eliminate  the  magnesium,  the  chlorine,  and  the  fluorine,  the  last,  accord- 
ing to  Gabriel,  occurring  only  as  traces,  the  remaining  mineral  bodies  form 
the  combination  3(Ca3P208)CaC03.  According  to  Gabriel  the  simplest 
expression  for  the  composition  of  the  ash  of  bones  and  teeth  is  (Ca3(P04)2  + 
Ca5HP30i3  + Aq),  in  which  2-3  per  cent  of  the  Ume  is  replaced  by  magnesia, 
potash,  and  soda,  and  4-6  per  cent  of  the  phosphoric  acid  by  carbonic  acid, 
chlorine,  and  fluorine. 

Analyses  of  bone-earths  have  shown  that  the  mineral  constituents  exist 
in  rather  constant  proportions,  which  are  nearly  the  same  in  different  animals. 
As  an  example  of  the  composition  of  bone-earth  we  here  give  the  analyses 
of  Zalesky.^    The  figures  represent  parts  per  thousand. 

Man.  Ox.  Tortoise.  Guinea-pig. 

Calcium  phosphate,  Ca3P,0g 838 . 9  860 . 9  859 . 8  873 . 8 

Magnesium  phosphate,  MgjPPg 10.4  10.2  13.6  10.5 

Calcium  combined  with  CO,,  Fl,  and  a...  76.5  73.6  63.2  70.3 

CO, " 57.3  62.0  52.7 

Chlorine 1.8  2.0         1.3 

Fluorine^ 2.3  3.0  2.0 

Some  of  the  CO2  is  always  lost  on  calcining,  so  that  the  bone-ash  does  not 
contain  the  entire  CO2  of  the  bony  substance. 

Ad.  Carnot  5  found  the  following  composition  for  the  bone-ash  of  man, 
ox,  and  elephant: 

Man.  Ox.  Elephant. 

&T  &.  Femur.  Femur. 

Calcium  phosphate 874.5  878.7  857.2  900.3 

Magnesium  phosphate 15.7  17.5  15.3  19.6 

Calcium  fluoride 3.5  3.7  4.5  4.7 

Calcium  chloride 2.3  3.0  3.0  2.0 

Calcium  carbonate 101.8  92.3  119.6  72.7 

Iron  oxide  .^ 1.0  1.3  1.3  1.5 

The  quantity  of  organic  substance  in  the  bones,  calculated  from  the  loss 
of  weight  in  burning,  varies  somewhat  between  300  and  520  p.  m.     This 

'  Zeitschr.  f.  phy.siol.  Chem.,  23. 

^Gabriel,  ibid.,  18,  which  also  contains  the  pertinent  literature;  Aron,  Pfliiger's 
Arch.,  106. 

'  Hojjpe-Seyler,  Med.-chem.  Untersuch.,  p.  19. 

''  The  statements  as  to  the  quantity  of  fluorine  are  contradictory;  see  Harms, 
Zeitschr.  f.  Biologic,  38;    Jodblauer,  ibid.,  41. 

*Compt.  rend.,  114. 


BONE.  437 

variation  may  in  part  be  explained  by  the  difficulty  in  obtaining  the  bony 
substance  entirely  free  from  water  and  partly  by  the  very  variable  amount 
of  blood-vessels,  nerves,  marrow,  and  the  like  in  different  bones.  The 
unequal  amounts  of  organic  substance  found  in  the  compact  and  in  the 
spongy  parts  of  the  same  bone,  as  well  as  in  bones  at  different  periods 
of  development  in  the  same  animal,  depend  probably  upon  the  varying 
quantities  of  these  above-mentioned  tissues.  Dentin,  which  is  compara- 
tively pure  bony  structure,  contains  only  260-280  p.  m.  organic  substance, 
and  Hoppe-Seyler  ^  therefore  thinks  it  probable  that  perfectly  pure  bony 
substance  has  a  constant  composition  and  contains  only  about  250  p.  m. 
organic  substance.  The  question  whether  these  substances  are  chemically 
combined  with  the  bone-earths  or  only  intimately  mixed  has  not  been 
decided. 

The  nutritive  fluids  which  circulate  through  the  bones  have  not  been  isolated, 
and  we  only  know  that  they  contain  some  protein  and  some  NaCl  and  alkali 
sulphate.  The  yellow  marrow  contains  chiefly  fat,  which  consists  of  olein,  pal- 
mitin,  and  stearin,  and  which  differs  from  the  fat  of  the  other  parts  of  the  body 
by  having  a  higher  acetyl  equivalent  (Zink  ^).  Protein  has  been  found  especially 
in  the  so-called  red  marrow  of  the  spongy  bones.  According  to  Forrest,  the 
protein  consists  of  a  globulin  coagulating  at  47-50°  C.  and  a  nucleoalbumin 
with  1.6  per  cent  phosphorus  (Halliburton  ^),  besides  traces  of  albumin.  Besides 
this  the  marrow  contains  so-called  extractive  bodies,  such  as  lactic  acid,  hypo- 
xanthine,  and  cholesterin,  but  mostly  bodies  of  an  unknown  character. 

The  diverse  quantitative  composition  of  the  various  bones  of  the  skele- 
ton depends  probably  on  the  varying  quantities  of  other  tissues,  such  as 
marrow,  blood-vessels,  etc.,  which  they  contain.  The  same  reason  explains, 
to  all  appearances,  the  larger  quantity  of  organic  substance  in  the  spongy 
parts  of  the  bones  as  compared  with  the  more  compact  parts.  Schrodt  ^ 
has  made  comparative  analyses  of  different  parts  of  the  skeleton  of  the 
same  animal  (dog)  and  has  found  an  essential  difference.  The  quantity  of 
water  in  the  fresh  bones  varies  between  138  and  443  p.  m.  The  bones  of 
the  extremities  and  the  skull  contain  138-222,  the  vertebrae  168-443,  and 
the  ribs  324-356  p.  m.  water.  The  quantity  of  fat  varies  between  13  and  269 
p.  m.  The  largest  amount  of  fat,  256-269  p.  m.,  is  found  in  the  long  tubular 
bones,  while  only  13-175  p.  m.  fat  is  found  in  the  small  short  bones.  The 
quantity  of  organic  substance,  calculated  from  fresh  bones,  was  150-300 
p.  m.,  and  the  quantity  of  mineral  substances  290-563  p.  m.  Contrary  to 
the  general  supposition  the  greatest  amount  of  bone-earths  was  not  found  in 
the  femur,  but  in  the  first  three  cervical  vertebras.  In  birds  the  tubular 
bones  are  richer  in  mineral  substances  than  the  fiat  bones  (DiJRiNG),  and 

'  Physiol.  Chem.,  102-104. 

2  See  Chem.  Centralbl.,  1897,  I,  2%. 

^  Forrest,  Journ.  of  Physiol.,  17  ;   Halliburton,  ibid.,  18. 

*  Cited  from  Maly's  Jahresber.,  (J. 


438  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

the  greatest  quantity  of  mineral  bodies  has  been  found  in  the  humerus 
(HiLLER,  During  i). 

We  do  not  possess  trustworthy  statements  in  regard  to  the  composition 
of  bones  at  different  ages.  The  analyses  by  E.  Voit  of  bones  of  dogs  and 
by  Brubacher  of  bones  of  children  apparently  indicate  that  the  skeleton 
becomes  poorer  in  water  and  richer  in  ash  with  increase  in  age.  Graffen- 
BERGER  ~  has  found  in  rabbits  6J-7J  years  old  that  the  bones  contained  only 
140-170  p.  m.  water,  while  the  bones  of  the  full-grown  rabbit  2-4  years  old 
contained  200-240  p.  m.  The  bones  of  old  rabbits  contain  more  carbon 
dioxide  and  less  calcium  phosphate. 

The  composition  of  bones  of  animals  of  different  species  is  but  little  known. 
The  bones  of  birds  contain,  as  a  rule,  somewhat  more  water  than  those  of  mam- 
malia, and  the  bones  of  fishes  contain  the  largest  quantity  of  water.  The  bones 
of  fishes  and  amphibians  contain  a  greater  amount  of  organic  substance.  The 
bones  of  pachyderm.s  and  cetaceans  contain  a  large  proportion  of  calcium  carbo- 
nate; those  of  granivorous  birds  always  contain  silicic  acid.  The  bone-ash  of 
amphibians  and  Tishes  contains  sodium  sulphate.  The  bones  of  fishes  seem  to 
contain  more  soluble  salts  than  the  bones  of  other  animals. 

A  great  many  experiments  have  been  made  to  determine  the  exchange  of 
material  in  the  bones — for  instance,  with  food  rich  in  lime  and  with  food 
deficient  in  lime — but  the  results  have  always  been  doubtful  or  contradic- 
tory. The  attempts,  also,  to  substitute  other  alkaline  earths  or  alumina  for 
the  lime  of  the  bones  have  given  contradictory  results.^  On  feeding  suffi- 
cient calcium  and  phosphorus  in  the  food  Aron  ^  found,  by  strongly  reducing 
the  sodium  and  at  the  same  time  giving  a  large  amount  of  potassium,  that 
the  development  of  the  bones  was  below  normal.  On  the  administration 
of  madder  the  bones  of  the  animal  are  found  to  be  colored  red  after  a  few 
days  or  weeks;  but  these  experiments  have  not  led  to  any  positive  con- 
clusion in  regard  to  the  growth  or  metabolism  in  the  bones. 

Under  pathological  conditions,  as  in  rachitis  and  softening  of  the  bones, 
an  ossein  has  been  found  which  does  not  give  any  typical  gelatine  on  boiling 
with  water.  Othen\'ise  pathological  conditions  seem  to  affect  chiefly  the 
quantitative  composition  of  the  bones,  and  especially  the  relationship 
between  the  organic  and  the  inorganic  substance.  In  exostosis  and  osteo- 
sclerosis the  quantity  of  organic  substance  is  generally  increased.  In 
rachitis  and  osteomalacia  the  quantity  of  bone-earths  is  considerably 
decreased.  Attempts  have  been  made  to  produce  rachitis  in  animals  by 
the  use  of  food  deficient  in  lime.     From  experiments  on  fully  developed 

^  Hiller,  cited  from  Maly's  Jahresber.,  14;   Diiring,  Zeitschr.  f.  physiol.  Chem.,  23. 
2  Voit,  Zeitschr.  f.   Biologie,  16;    Brubacher,  ibid.,  27;    Graffenberger  in  Maly's 
Jahresber.,  21. 

^  See  H.  Weiske,  Zeitschr.  f.  Biologie,  31. 
*  Pfl'ger's  Arch.,  lOG. 


BONE.  439 

animals  contradictor}'-  results  have  been  obtained.  In  young,  undeveloped 
.animals  Erwix  Voit  ^  produced,  by  lack  of  lime-salts,  a  change  similar  to 
rachitis.  In  fuU-growTi  animals,  the  bones  were  changed  after  a  long  time 
because  of  the  lack  of  lime-salts  in  the  food,  but  did  not  become  soft,  only 
thinner  (osteoporosis).  The  attempts  to  remove  the  lime-salts  from  the 
bones  by  the  addition  of  lactic  acid  to  the  food  have  led  to  no  positive 
results  (Heitzmaxn,  Heiss,  Bagixsky^).  Weiske,  on  the  contrary,  has 
shown,  by  administering  dilute  sulphuric  acid  or  monosodium  phosphate 
with  the  food  (presupposing  that  the  food  gave  no  alkaline  ash)  to  sheep 
and  rabbits,  that  the  quantity  of  mineral  bodies  in  the  bones  might  be 
diminished.  On  feeding  continuously  for  a  long  time  with  a  food  which 
yielded  an  acid  ash  (cereal  grains),  Weiske  has  observed  a  diminution  in 
the  mineral  substances  of  the  bones  in  full-grown  herbivora.^  A  few  inves- 
tigators are  of  the  opinion  that  in  rachitis,  as  in  osteomalacia,  a  solution 
of  the  lime-salts  by  means  of  lactic  acid  takes  place.  This  was  suggested 
by  the  fact  that  O.  Weber  and  C.  Schmidt  •*  found  lactic  acid  in  the  cyst- 
like, altered  bony  substance  in  osteomalacia. 

Well-known  investigators  have  disputed  the  possibility  of  the  lime- 
salts  being  washed  from  the  bones  in  osteomalacia  by  means  of  lactic 
acid.  They  have  given  special  prominence  to  the  fact  that  the  lime-salts 
held  in  solution  by  the  lactic  acid  must  be  deposited  on  neutralization  of 
the  acid  by  the  alkaline  blood.  This  objection  is  not  very  important,  as 
the  alkaline  blood-serum  has  the  property  to  a  high  degree  of  holding  earthy 
phosphates  in  solution,  which  fact  can  be  easily  proved.  The  investiga- 
tions of  Levy  ^  contradict  the  statement  as  to  the  solution  of  the  lime-salts 
by  lactic  acid  in  osteomalacia.  He  has  found  that  the  normal  relationship 
6PO4:10Ca  is  retained  in  all  parts  of  the  bones  in  osteomalacia,  which 
would  not  be  the  case  if  the  bone-earths  were  dissolved  by  an  acid.  The 
decrease  in  phosphate  occiu^  in  the  same  quantitative  relationship  as  the 
carbonate,  and  according  to  Le\"y,  in  osteomalacia  the  exhaustion  of  the 
bone  takes  place  by  a  decalcification  in  which  one  molecule  of  phosphate 
carbonate  after  the  other  is  removed. 

In  rachitis  the  quantity  of  organic  matter  has  been  found  to  vary  between  664 
and  811  p.  m.  The  quantity  of  inorganic  substance  was  189-336  p.  m.  These 
figiu'es  refer  to  the  dried  substance.  According  to  Brubacher,  rachitic  bones 
are  richer  in  water  than  the  bones  of  healthy  childi'en,  and  poorer  in  mineral 

1  Zeitschr.  f.  Biologie,  16. 

^  Heitzniann,  Maly's  Jahresber.,  3,  229;  Heiss,  Zeitschr.  f.  Biologie,  12;  Baginsky, 
Virchow's  Arch.,  87. 

'  See  Maly's  Jahresber.,  22;  also  Weiske.  Zeitschr.  f.  physiol.  Chem.,  20,  and 
Zeitschr.  f.  Biologie,  31. 

*  Cited  from  v.  Gorup-Besanez,  Lehrh.  d.  physiol.  Chem.,  4.  Aufl. 

*  Zeitschr.  f.  physiol.  Chem.,  19. 


440  TISSUES   OF  THE   CONNECTIVE    SUBSTANCE. 

bodies,  especially  calcium  phosphate.  In  opposition  to  rachitis,  osteomalacia 
is  often  characterized  by  the  considerable  amount  of  fat  in  the  bones,  230-290 
p.  m.;  but  as  a  rule  the  composition  varies  so  much  that  the  analyses  are  of  little 
value.  In  a  case  of  osteomalacia,  Chabrie  '  found  a  larger  quantity  of  magne- 
sium than  calcium  in  a  bone.  The  ash  contained  417  p.  m.  phosphoric  acid,  222 
p.  m.  lime,  269  p  m.  magnesia,  and  86  p.  m.  carbon  dioxide.  Other  investigators 
have  on  the  contrary  found  considerably  more  calcium  than  magnesium. 

The  tooth-structure  is  nearly  related,  from  a  chemical  standpoint,  to 
the  bony  structure. 

Of  the  three  chief  constituents  of  the  teeth — dentin,  enamel,  and 
cement — the  cement  is  to  be  considered  as  true  bony  structure,  and  as 
such  has  already  been  discussed  to  some  extent.  Dentin  has  the  same 
composition  as  the  bony  structure,  but  contains  somewhat  less  water.  The 
organic  substance  yields  gelatine  on  boiling;  but  the  dental  tubes  are 
not  dissolved,  therefore  they  cannot  consist  of  collagen.  In  dentin  260-280 
p.  m.  organic  substance  has  been  found.  Enamel  is  an  epithelium  forma- 
tion containing  a  large  proportion  of  lime-salts.  Corresponding  to  its 
character  and  origin,  the  organic  substance  of  the  enamel  does  not  yield 
any  gelatine.  Completely  developed  enamel  contains  the  least  water,  the 
greatest  quantity  of  mineral  substances,  and  is  the  hardest  of  all  the  tissues 
of  the  body.  In  full-grown  animals  it  contains  hardly  any  water,  and  the 
quantity  of  organic  substance  amounts  to  only  20-40-68  p.  m.  The  rela- 
tive amounts  of  calcium  and  phosphoric  acid  are,  according  to  the  analyses 
of  Hoppe-Seyler,  about  the  same  as  in  bone-earths.  The  quantity  of 
chlorine  according  to  Hoppe-Seyler  is  remarkably  high,  0.3-0.5  per  cent, 
while  Bertz^  found  that  the  ash  of  enamel  was  free  from  chlorine  and 
that  dentin  was  very  poor  in  chlorine. 

Carnot,^  who  has  investigated  the  dentin  from  elephants,  has  found  4.3  p.  m. 
calcium  fluoride  in  the  ash.  In  ivory  he  found  only  2  p.  m.  Dentin  from 
elephants  is  rich  in  magnesium  phosphate,  which  is  still  more  abundant  in  ivory. 

According  to  Gabriel  the  amount  of  fluorine  is  very  small  and  amounts 
to  1  p.  m.  in  ox-teeth.  It  is  no  greater  in  the  teeth  and  enamel  than  in 
the  bones.*  The  same  investigator  found  that  the  amount  of  phosphates  is 
strikingly  small  in  the  enamel,  and  in  the  teeth  considerable  lime  is 
replaced  by  magnesia.  This  coincides  with  Bertz's  findings,  that  dentin 
contains  twice  as  much  magnesia  as  the  enamel. 

'  Chabrie,  Les  phenomenes  cliim.  de  rossification,  Paris,  1895, 65. 

^  See  Maly's  Jahresber.,  30. 

^  Compt.  rend.,  114. 

*  See  foot-note  4,  p.  436. 


FATTY  TISSUE.  441 


IV.    The  Fatty  Tissue. 

The  membranes  of  the  fat-cells  withstand  the  action  of  alcohol  and 
ether.  They  are  not  dissolved  by  acetic  acid  nor  by  dilute  mineral  acids, 
but  are  dissolved  by  artificial  gastric  juice.  They  may  possibly  consist  of  a 
substance  closely  related  to  elastin.  The  fat-cells  contain,  besides  fat,  a 
yellow  pigment  which  in  emaciation  does  not  disappear  so  rapidly  as  the 
fat;  and  this  is  the  reason  that  the  subcutaneous  cellular  tissue  of  an 
emaciated  corpse  has  a  dark  orange-red  color.  The  cells  deficient  in  or 
nearly  free  from  fat,  which  remain  after  the  complete  disappearance  of  the 
latter,  seem  to  have  an  albuminous  protoplasm  rich  in  water.  Adipose 
tissue  is  rich  in  a  fat-splitting  enzyme  and  in  catalases  (see  Chapter  I). 

The  less  water  the  fatty  tissue  contains  the  richer  it  is  in  fat.  Schulze 
and  Reixecke  ^  found  in  1000  parts 

Water.  Membrane.  Fat. 

Fatty  tissue  of  oxen 99.7  16.6  883.7 

"      "sheep 104.8  16.4  878.8 

"      "pigs 64.4  13.6  922.0 

The  fat  contained  in  the  fat-cells  consists  chiefly  of  triglycerides  of 
stearic,  palmitic,  and  oleic  acids.  Besides  these,  especially  in  the  less  solid 
kinds  of  fats,  there  are  glycerides  of  other  fatty  acids  (see  Chapter  IV). 
In  all  animal  fats  there  are  besides  these,  as  Fr.  Hofmaxx  -  has  shoT\Ti, 
also  free,  non-volatile  fatty  acids,  although  in  very  small  amounts. 

Human  fat  is  relatively  rich  in  olein,  the  quantity  in  the  subcutaneous 
fatty  tissue  being  70-80  per  cent  or  more.^  In  new-bom  infants  it  is  poorer 
in  oleic  acid  than  in  adults  (Kxopfelila.cher,  Siegert,  Jaeckle);  the 
quantity  of  olein  increases  until  the  end  of  the  first  year,  when  it  is  about 
the  same  as  in  adults.  The  composition  of  the  fat  in  man  as  well  as  in 
different  incUviduals  of  the  same  species  of  animals  is  rather  variable,  a 
fact  which  is  probably  dependent  upon  the  food.  AccorcUng  to  the 
researches  of  Hexriques  and  Haxsex  the  fat  of  the  subcutaneous  fatty 
tissue  is  richer  in  .olein  than  that  of  the  internal  organs;  this  has  also  been 
observed  by  Leick  and  Wixkler.*  In  animals  with  a  thick  subcutaneous 
fat  deposit  the  outer  layers,  according  to  Hexriques  and  Haxsex'^,  are 
richer  in  olein  than  the  inner  layers.  The  fat  of  cold-blooded  arimals  is 
especially  rich  in  olein.     The  fat  of  domestic  animals  has.  according  to 

*  Annal.  d.  Cliem.  u.  Pharm..  142. 

^  Ludwig-Festschrift,  1874,  Leipzig. 

'  See  Jaeckle,  Zeitschr.  f.  physiol.  Chem.,  36  (literature). 

*  Knopfelmacher,  Jahrbuch  f.  Kinderheilkunde  (X.  F.),  45  (older  literature); 
Siegert.  Hofmeister's  Beitrage,  1;  Jaeckle.  Zeitschr.  f.  physiol.  Chem.,  36  (literature); 
Henriques  and  Hansen,  Skand.  Arch.  f.  Physiol..  11;  Leick  and  Winkler.  .\rch.  f. 
Path.  u.  Pharm.,  48. 


442  TISSUES   OF   THE    CONNECTIVE    SUBSTANCE. 

Amthor  and  Zink,  a  less  oily  consistency  and  a  lower  iodine  and  acetyl 
equivalent  than  the  corresponding  fat  of  wild  animals.  Under  pathological 
conditions  the  fat  may  have  a  markedly  pronounced  variation.  The  fat 
of  lipoma  seems,  according  to  Jaeckle,  to  be  poorer  in  lecithin  than  other 
fats. 

The  properties  of  fats  in  general,  and  the  three  most  important  varieties 
of  fat,  have  already  been  considered  in  a  previous  chapter,  hence  the  forma- 
tion of  the  adipose  tissue  is  of  chief  interest  at  this  time. 

The  formation  of  fat  in  the  organism  may  occur  in  various  ways.  The 
fat  of  the  animal  body  may  consist  partly  of  fat  absorbed  from  the  food 
and  deposited  in  the  tissues,  and  partly  of  fat  formed  in  the  organism 
from  other  bodies,  such  as  proteins  or  carbohydrates. 

That  the  fat  from  the  food  which  is  absorbed  in  the  intestinal  canal  may 
be  retained  by  the  tissues  has  been  shown  in  several  ways.  Radziejewski, 
Lebedeff,  and  Munk  have  fed  dogs  with  various  fats,  such  as  linseed-oil, 
mutton-tallow,  and  rape-seed-oil,  and  have  afterwards  found  the  adminis- 
tered fat  in  the  tissues.  Hofmann  starved  dogs  until  they  appeared  to 
have  lost  their  fat  and  then  fed  them  upon  large  quantities  of  fat  and  only 
little  proteins.  When  the  animals  were  killed,  he  found  so  large  a  quantity 
of  fat  that  it  could  not  have  been  formed  from  the  administered  proteins 
alone,  but  the  greater  part  must  have  been  derived  from  the  fat  of  the 
food.  Pettenkofer  and  Voit  arrived  at  similar  results  in  regard  to  the 
behavior  of  the  absorbed  fats  in  the  organism,  though  their  experiments 
were  of  another  kind.  Munk  has  found  that  on  feeding  with  free  fatty 
acids,  these  are  deposited  in  the  tissues,  not,  however,  as  such;  but  they 
are  transformed  by  synthesis  with  glycerine  into  neutral  fats  on  their  pas- 
sage from  the  intestine  into  the  thoracic  duct.  The  connection  between 
the  fat  of  the  food  and  of  the  body  has  also  been  shown  by  oth*^rs,  espe- 
cially by  Rosenfeld.  Coroxedi  and  Marchetti  and  especially  Winter- 
NiTZ  1  have  recently  shown  that  the  iodized  fat  is  taken  up  in  the  intestinal 
tract  and  deposited  in  the  various  organs. 

Proteins  and  carbohydrates  are  considered  as  the  mother-substances  of 
the  fats  formed  in  the  organism. 

The  formation  of  the  so-called  corpse-wax,  adipocere,  which  consists  of 
a  mixture  of  fatty  acids,  ammonia,  and  lime-soaps,  from  parts  of  the  corpse 
rich  in  proteins,  is  sometimes  given  as  a  proof  of  the  formation  of  fats  from 
proteins.  The  accuracy  of  this  view  has,  however,  been  disputed,  and 
many  other  explanations  of  the  formation  of  this  substance  have  been 
offered.     According  to  the  experiments  of  Kratter  and  K.  B.  Lehmann, 

'  Coronedi  and  Marchetti,  cited  by  Winternitz,  Zeitschr.  f.  physiol.  Chem.,  24. 
A  review  of  the  literature  on  fat  formation  may  be  found  in  Rosenfeld,  Fettbildung, 
in  Ergebnisse  der  Physiologic,  1,  Abt.  1. 


FORMATION  OF  FAT.  443 

it  seems  as  if  it  were  possible  by  experimental  means  to  convert  animal 
tissue  rich  in  proteins  (muscles)  into  adipocere  by  the  continuous  action 
of  water.  Irrespective  of  this,  Salkowski  has  shown  recently  that  in 
the  formation  of  adipocere  the  fat  itself  takes  part,  in  that  tlie  olein  decom- 
poses with  the  formation  of  solid  fatty  acids;  still  it  must  be  considered 
that  lower  organisms  undoubtedly  take  part  in  its  formation.  The  pro- 
duction of  adipocere  as  a  proof  of  the  formation  of  fat  from  proteins  is 
disputed  by  many  investigators  for  this  and  other  reasons. 

Fatty  degeneration  has  been  considered  as  another  proof  of  the  forma- 
tion of  fat  from  proteins.  From  the  investigations  of  Bauer  on  dogs  and 
Leo  on  frogs  it  was  assumed  that,  at  least  in  acute  poisoning  by  phos- 
phorus, a  fatty  degeneration,  with  the  formation  of  fat  from  proteins, 
takes  place.  Pfluger  has  raised  such  strong  arguments  against  the  older 
researches  as  well  as  the  more  recent  one  of  Polimaxti,  who  claims  to 
have  sho-uTi  the  formation  of  fat  from  proteins  in  phosphorus  poisoning, 
that  we  cannot  consider  the  formation  of  fat  as  conclusively  proved. 
Recent  investigations  of  Athaxasiu.  Taylor.  Schwalbe,  and  others, 
especially  of  Rosexfeld.^  have  made  it  probable  that  in  these  instances  no 
new  formation  of  fat  from  protein  took  place,  but  rather  a  fat  migration 
(Rosexfeld). 

Another  more  direct  proof  for  the  formation  of  fat  from  proteins  has 
been  given  by  Hofmaxx.  He  experimented  with  fly-maggots.  A  num- 
ber of  these  were  killed  and  the  quantity  of  fat  determined.  The  remainder 
were  allowed  to  develop  in  blood  whose  proportion  of  fat  had  been  pr3\'i- 
ously  determined,  and  after  a  certain  time  they  were  killed  and  analyzed. 
He  found  in  them  from  seven  to  eleven  times  as  much  fat  as  was  contained 
in  the  maggots  first  analyzed  and  the  blood  taken  together.  Pfluger-  has 
made  the  objection  that  a  considerable  number  of  lower  fungi  develop  in 
the  blood  under  these  conditions,  in  whose  cell-body  fats  and  carbohydrates 
are  formed  from  the  different  constituents  of  the  blood  and  their  decompo- 
sition products,  and  that  these  serve  as  food  for  the  maggots. 

As  a  more  convincing  proof  of  fat  formation  from  proteins,  the  investi- 
gations of  Pettexkofer  and  Voit  are  often  quoted.  These  investigators 
fed  dogs  with  large  quantities  of  meat  containing  the  least  possible  propor- 
tion of  fat,  and  found  all  of  the  nitrogen  in  the  excreta,  but  only  a  part  of 
the  carbon.  As  an  explanation  of  these  conditions  it  has  been  assumed  that 
the  protein  of  the  organism  splits  into  a  nitrogenized  and  a  non-nitrog- 
enized   part,   the   former   changing   into   the   nitrogenized   final    product, 

'Bauer.  Zeitschr.  f.  Biologie,  7;  Leo,  Zeitschr.  f.  physiol.  Chem.,  9;  Polimanti, 
Pfl  ger's  Arch.,  70;  Pfluger.  ibid..  51  (literature  on  the  formation  of  fat  from  protein) 
and  71;    Athanasiu,  ibid.,  7-t;   Taylor,  Joum.  Exp.  Medicine,  -l;   see  also  foot-note  1, 


Oi 


See  Rosenfeld,  Fettbildung,  Ergebnisse  der  Physiologie,  1,  Abt.  1. 


444  TISSUES   OF  THE  CONNECTIVE    SUBSTANCE. 

urea,  and  like  products,  and  the  latter,  on  the  contrary,  being  retained 
in  the  organism  as  fat  (Pettenkofer  and  Voit). 

Pfluger  has  arrived  at  the  following  conclusion  by  an  exhaustive 
criticism  of  Pettenkofer  and  Voit's  experiments  and  a  careful  recal- 
culation of  their  balance-sheet:  that  these  very  meritorious  investigations, 
which  vrere  continued  for  a  series  of  years,  were  subject  to  such  great 
defects  that  they  are  not  conclusive  as  to  the  formation  of  fat  from  pro- 
teins. He  especially  emphasizes  the  fact  that  these  investigators  started 
from  a  wrong  assumption  as  to  the  elementary  composition  of  the  meat, 
and  that  the  quantity  of  nitrogen  assumed  by  them  was  too  low  and  the 
quantity  of  carbon  too  high.  The  relationship  of  nitrogen  to  carbon  in 
meat  poor  in  fat  was  assumed  by  Voit  to  be  as  1:  3.68,  while  according  to 
PFLtJGER  it  is  1 ;  3.22  for  fat-free  meat  after  deducting  the  glycogen,  and 
according  to  Rubner  1,3.28  without  deducting  the  glycogen.  On  recalcu- 
lation of  the  figures  using  these  coefficients,  Pfutger  has  arrived  at  the 
conclusion  that  the  assumption  as  to  the  formation  of  fat  from  proteins 
finds  no  support  in  these  experiments. 

In  opposition  to  these  objections,  E.  Voit  and  M.  Cremer  have  made 
new  feeding  experiments  to  show  the  formation  of  fat  from  proteins,  but 
the  proof  of  these  recent  investigations  has  been  denied  by  Pfluger.  On 
feeding  a  dog  on  meat  poor  in  fat  (containing  a  known  quantity  of  ether 
extractives,  glycogen,  nitrogen,  water,  and  ash),  Kumagawa^  could  not 
prove  the  formation  of  fat  from  protein.  According  to  him  the  animal 
body  under  normal  conditions  has  not  the  power  of  forming  fat  from  pro> 
tein. 

Several  French  investigators,  especially  Chauveau,  Gautier,  and 
Kaufmann,  consider  the  formation  of  fat  from  proteins  as  positively  proved. 
Katjfmann  has  recently  substantiated  this  view  b}^  a  method  which  will 
be  spoken  of  in  detail  in  Chapter  XVIII,  in  which  he  studied  the  nitro- 
gen elimination  and  the  respiratory  gas  exchange  in  conjunction  with  the 
simultaneous  formation  of  heat. 

As  we  are  agreed  that  carbohydrates  and  glycogen,  as  well  as  sugar, 
can  be  formed  from  proteins,  the  fact  cannot  be  denied  that  possibly  an 
indirect  formation  of  fat  from  proteins,  with  a  carbohydrate  as  an  inter- 
mediate step,  can  take  place.  The  possibility  of  a  direct  fat  formation 
from  proteins  without  the  carbohydrate  as  intermediary  must  also  be 
generally  admitted,  although  such  a  formation  has  not  been  conclusively 
proved. 

According  to  Chauveau  and  Kaufmann,  in  the  direct  formation  of  fat 

*  See  Rosenfeld,  Fettbildung,  Ergebnisse  der  Physiologie,  1,  Abt.  ]. 

*  Kaufmann,  Arch,  de  physiol.  (5)    8,  where  the  worLs  of  Chauveau  and  Gautier 
are  cited. 


FORMATION   OF  FAT.  445 

from  proteins  the  fat  is  formed,  besides  urea,  carbon  dioxide,  and  water, 
as  an  intermediary  product  in  the  oxidation  of  the  proteins,  while  Gautier 
considers  the  formation  of  fat  from  proteins  as  a  cleavage  without  the  tak- 
ing up  of  oxygen.  If  fat  is  formed  from  protein  in  the  animal  body,  then 
such  formation  is  not  a  splitting  off  of  fat  from  the  proteins,  but  rather  a 
synthesis  from  primarily  formed  cleavage  products  of  proteins  which 
are  deficient  in  carbon. 

The  formation  oj  jot  from  carbohydrates  in  the  animal  body  was  first 
suggested  by  Liebig.  This  was  combated  for  some  time,  and  until  lately  it 
was  the  general  opinion  that  a  direct  formation  of  fat  from  carbohydrates  not 
only  had  not  been  proved,  but  also  that  it  was  improbable.  The  imdoubt- 
edly  great  influence  of  the  carbohydrates  on  the  formation  of  fat  as  ob- 
served and  proved  by  Liebig  was  explained  by  the  statement  that  the 
carbohydrates  were  consumed  instead  of  the  absorbed  fat  or  that  derived 
from  the  proteins,  hence  they  have  a  sparing  action  on  the  fat.  Bv  means  of 
a  series  of  nutrition  experiments  with  foods  especially  rich  in  carbohydrates 
Lawes  and  Gilbert,  Soxhlet,  Tscherwinsky,  Meissl  and  Stromer  (on 
pigs),  B.  Schultze,  Chaniew^ski,  E.  Voit  and  C.  Lehmaxn  (on  geese),  I. 
MuNK  and  Rubner  and  Lummert  ^  (on  dogs)  apparently  prove  that  a 
direct  formation  of  fat  from  carbohydrates  does  actually  occur.  The 
processes  by  which  this  formation  takes  place  are  still  unknown.  As  the 
carbohydrates  do  not  contain  as  complicated  carbon  chains  as  the  fats,  the 
formation  of  fat  from  carbohydrates  must  consist  of  a  synthesis,  in  which 
the  group  CHOH  is  converted  into  CHo;  hence  a  reduction  must  occur. 

Analogous  to  Nexcki's  view  as  to  the  butyric-acid  fermentation,  when 
lactic  acid  is  formed  from  the  sugar  and  from  this  CO2H2  and  acetaldeliyde 
(C2H4O)  are  produced,  and  from  this  latter,  by  the  union  of  two  molecules, 
butyric  acid  is  formed,  so  Magnus-Levy  2  attempts  to  explain  the  forma- 
tion of  fat  in  the  animal  body  from  carbohydrates  by  synthesis  from 
aldehyde  and  reduction.  He  considers  that  the  process  proceeds  in  the 
following  way:  (a)  9C3H603  =  9C2H40  +  9H2  +  9C02  and  (6)  9C2H4O  +  7H2 
=  CisH3602  (stearic  acid) +7H2O. 

After  feeding  with  very  large  quantities  of  carbohydrates  the  relation- 
ship between  the  inspired  oxygen  and  the  expired  carbon  dioxide,  i.e.,  the 

CO 
respiratoiy  quotient  -j^,  was  found  greater  than  1  in  certain  cases  (Han- 

'  Lawes  and  Gilbert,  Phil.  Transactions,  1859,  part  2;  Soxhlet,  see  ^laly's  Jahresber., 
11,  51;  Tscherwinsky,  Landwirthsch.  Versuchsstaat,  29  (cited  from  Maly's  Jahresber., 
13);  Meissl  and  Stromer,  Wien.  Sitzungsber.,  SS,  Abt.  3;  Schultze,  Maly's  -Jahresber., 
11,  47;  Chaniewski,  Zeitschr  f.  Biologic,  20;  Voit  and  Lehmann,  see  C.  v  Voit,  Sitz- 
ungsber,  d.  k.  bayer  Akad.  d.  Wissensch.,  1885;  I.  Munk.  Virchow's  Arch.,  101; 
Rubner,  Zeitschr  f.  Biologic,  22;   Lummert,- Pfliiger's  Arch..  "1. 

•  Arch.  f.  (Anat.  u.)  Physiol.,  1901. 


446  TISSUES  01    THE  CONNECTIVE  SUBSTANCE. 

RIOT  and  RiCHET,  Bleibtreu,  Kaufmann,  Laulanie  ^).  This  is  explained 
by  the  assumption  that  the  fat  is  formed  from  the  carbohydrate  by  a 
cleavage  setting  free  carbon  dioxide  and  water  without  taking  up  oxgen. 
This  increase  in  the  respiratory  quotient  also  depends  in  part  on  the 
increased  combustion  of  the  carbohydrate. 

When  food  contains  an  excess  of  fat  the  superfluous  amount  is  stored 
up  in  the  fatty  tissue,  and  on  partaking  of  food  deficient  in  fat  this  accu- 
mulation is  quickly  exliausted;  and  it  is  very  probable  that  the  lipase  is 
of  importance  here,  as  Loevenhart^  has  found  that  all  over  the  body 
where  fat  is  deposited  in  large  amounts  lipase  also  occurs  in  considerable 
amounts.  There  is  perhaps  not  one  of  the  various  tissues  that  decreases 
so  much  in  starvation  as  the  fatty  tissue.  The  organism,  then,  possesses 
in  this  tissue  a  depot  where  there  is  stored  during  proper  alimentation  a 
nutritive  substance  of  great  importance  in  the  development  of  heat  and 
vital  force,  which  substance,  on  insufficient  nutrition,  is  given  up  as  may 
be  needed.  On  account  of  their  low  conducting  power,  the  fatty  tissues 
become  of  great  importance  in  regulating  the  loss  of  heat  from  the  body. 
They  also  serve  to  fill  cavities  and  act  as  a  protection  and  support  to 
certain  internal  organs. 

'  Hanriot  and  Riehet,  Annal.  de  Chim.  et  de  Phys.  (6),  23;    Bleibtreu,  Pfliiger's 
Arch.,  56  and  85;  Kaufmann,  Arch,  de  Physiol.  (5),  8;  Laxilanie,  ibid.,  791. 
^  Amer.  Journ.  of  Physiol.,  6. 


CHAPTER  XI. 
MUSCLES. 

Striated  Muscles. 

In  the  study  of  the  muscles  the  chief  problem  for  physiological  chem- 
istry is  to  isolate  their  different  morphological  elements  and  to  investigate 
each  element  separately.  By  reason  of  the  complicated  structure  of  the 
muscles  this  has  been  thus  far  almost  impossible,  and  we  must  be  satisfied 
at  the  present  time  with  a  few  microchemical  reactions  in  the  investi- 
gation of  the  chemical  composition  of  the  muscular  fibres. 

Each  muscle-tube  and  each  muscle-fibre  consists  of  a  sheath,  the  sar- 
COLEMMA,  which  scems  to  be  composed  of  a  substance  similar  to  elastin, 
and  containing  a  large  proportion  of  'protein.  This  last,  which  in  life  pos- 
sesses the  power  of  contractility,  has  in  the  inactive  muscle  an  alkaline 
reaction,  or,  more  correctly  speaking,  an  amphoteric  reaction  with  a  pre 
dominating  action  on  red  litmus  paper.  Rohmann  has  found  that  the 
fresh,  inactive  muscle  shows  an  alkaline  reaction  with  red  lacmoid,  and  an 
acid  reaction  with  brown  turmeric.  From  the  behavior  of  these  coloring- 
matters  with  various  acids  and  salts  he  concludes  that  the  alkalinity  of 
the  fresh  muscle  with  lacmoid  is  due  to  sodium  bicarbonate,  diphosphate, 
and  probably  also  to  an  alkaline  combination  of  protein  bodies,  and  the 
acid  reaction  with  turmeric,  on  the  contrary,  to  monophosphate  chiefly. 
The  dead  muscle  has  an  acid  reaction,  or,  more  correctly,  the  acidity  with 
turmeric  increases  on  the  decease  of  the  muscle,  and  the  alkalinity  with 
lacmoid  decreases.  The  difference  depends  on  the  presence  of  a  larger 
quantity  of  monophosphate  in  the  dead  muscle,  and  according  to  Rohmann 
free  lactic  acid  is  found  in  neither  the  one  case  nor  the  other.! 

If  the  somewhat  disputed  statements  relative  to  the  finer  structure  of 
the  muscles  are  disregarded,  one  can  differentiate  in  the  striated  muscles 
between  the  two  chief  components,  the  doubly  refracting — anisotropous — 
and  the  singly  refracting — isotropous — substance.  If  the  muscular  fibres 
are  treated  with  reagents  which  dissolve  proteins,  such  as  dilute  hydro- 

'  The  various  statements  in  regard  to  the  reaction  of  the  muscles  and  the  cause 
thereof  are  conflicting.  See  Rohmann,  Pfli  ger's  Arch.,  50  and  Ho;  Heffter,  Arch.  f. 
exp.  Path.  u.  Pharm.,  31  and  38.     These  references  contain  the  pertinent  literature. 

447 


448  MUSCLES. 

chloric  acid,  soda  solution,  or  gastric  juice,  they  swell  greatly  and  break 
up  into  "Bowal^n's  disks."  B}^  the  action  of  alcohol,  chromic  acid, 
boiling  water,  or  m  general  such  reagents  as  cause  a  shrinking,  the  fibres 
split  longitudinally  into  fibrils;  and  this  behax-ior  shows  that  several 
chemically  different  substances  of  various  solubilities  enter  into  the  con- 
struction of  the  muscular  fibres. 

The  protein  mj'osin  is  generally  considered  as  the  chief  constituent  of 
the  diagonal  disks,  while  the  isotropous  substance  contains  the  chief  mass 
of  the  other  proteins  of  the  muscles  as  well  as  the  chief  portion  of  the 
extractives.  According  to  the  observations  of  Daxilew^sky,  confirmed  by 
J.  HoLMGREx,!  mvosin  may  be  completely  extracted  from  the  muscle  with- 
out changing  its  structure,  by  means  of  a  5  per  cent  solution  of  ammonium 
chloride,  which  fact  contradicts  the  above  view.  Danilewsky  claims  that 
another  protein-like  substance,  insoluble  in  ammonium  chloride  and  only 
swelling  up  therein,  enters  essentially  into  the  structure  of  the  muscles. 
The  proteins,  which  form  the  chief  part  of  the  solids  of  the  muscles,  are  of 
the  greatest  importance. 

Proteins  of  the  Muscles. 

Like  the  blood  which  contams  a  fluid,  the  blood-plasma,  which  sponta- 
neously coagulates,  separating  fibrin  and  yielding  blood-serum,  so  also  the 
living  muscle,  at  least  of  cold-blooded  animals,  contains,  as  first  shown  by 
KiJHXE,  a  spontaneously  coagulating  liquid,  the  muscle-plasma,  which 
coagulates  quickly,  separating  a  protein  body,  myosin,  and  yielding  also 
a  serum.  That  liquid  which  is  obtained  by  pressing  the  living  muscle  is 
called  jnuscle-plasma,  while  that  obtained  from  the  dead  muscle  is  called 
muscle-seruryi.    These  two  fluids  contain  different  protein  bodies. 

Muscle-plasma  was  first  prepared  by  Kuhxe  from  frog-muscles,  and 
later  by  Halliburton,  according  to  the  same  method,  from  the  muscles 
of  warm-blooded  animals,  especially  rabbits.  The  principle  of  this  method 
is  as  follows:  The  blood  is  removed  from  the  muscles  immediately  after 
the  death  of  the  animal  by  passing  through  them  a  strongly  cooled  com- 
mon-salt solution  of  5-6  p.  m.  Then  the  muscles  are  quickly  cut  and 
immediately  thoroughly  frozen  so  that  they  can  be  groimd  in  this  state 
to  a  fine  mass —  "muscle-snow."  This  pulp  is  strongly  pressed  in  the  cold, 
and  the  liquid  which  exudes  is  called  muscle-plasma.  According  to  v. 
FuRTH  2  this  cooliug  or  freezing  is  not  necessary.     It  is  sufficient  to  extract 


1  Danilewsky,  Zeitschr.  f.  physiol.  Chem.,  7;   J.  Holmgren,  ]\Ialy's  Jahresber.,  23. 

2  See  Krhne,  Untersuchungen  i.ber  das  Protoplasma  (Leipzig,  1864),  2;  Hallibur- 
ton, Journ.  of  Physiol.,  8;  v.  Furth,  Arch.  f.  exp.  Path.  u.  Pharm.,  36  and  3";  Hof- 
meister's  Beitrage,  3,  and  Ergebuisse  der  Physiologic,  1,  Abt.  1;  Stewart  and  Soll- 
mann,  Journ.  cf  Physiol.,  24. 


PROTEINS  OF  THE  MUSCLE.  449 

the  muscle  free  from  blood,  as  above  directed,  with  a  6  p.  m.  common- 
salt  solution. 

Muscle-plasma  forms  a  yellow  to  brownish-colored  fluid  with  an  alkaline 
reaction.  It  is  somew^hat  different  in  different  animals.  Muscle-plasma 
from  the  frog  spontaneously  coagulates  slowly  at  a  little  above  0°C.,  but 
more  quickly  at  the  temperature  of  the  body.  Muscle-plasma  from  mammals 
coagulates  slowly,  according  to  v.  FtJRTH,  even  at  the  temperature  of  the 
room,  though  only  slightly,  and  it  can  hardly  be  considered  as  a  process 
comparable  with  the  coagulation  of  the  blood.  Indeed  the  question  may  be 
asked  whether  a  true  muscle-plasma  does  exist  in  warm-blooded  animals, 
or  whether  the  fluid  obtained  from  such  muscles  exactly  represents  the 
plasma  of  the  living  muscle.  According  to  Kuhxe  and  v.  Furth  the  reac- 
tion remains  alkaline  during  coagulation,  while  according  to  Halliburtox, 
Stewart  and  Soll.maxx,  it  becomes  acid.  According  to  the  older  \dews 
the  clot  consists  of  a  globulin  called  myosin,  while  v.  Furth  claims  that  it 
consists  of  two  coagulated  proteins,  myosin-fibrin  and  myogen-fibrin. 

The  study  of  the  proteins  of  the  muscles,  as  well  as  their  nomenclature, 
has  changed  markedly  in  the  last  few  years,  and  it  is  questionable  whether 
an  essential  difference  exists  between  the  proteins  of  the  muscle-plasma 
and  the  muscle-serum  of  warm-blooded  animals.  Nevertheless  it  is  neces- 
sar}^  to  separately  discuss  the  proteins  of  the  dead  muscle  as  well  as  those 
of  the  muscle-plasma. 

The  proteins  of  the  dead  muscle  are  in  part  soluble  in  water  or  dilute 
salt  solutions,  and  in  part  are  insoluble  therein.  Myosin  and  musculin  and 
also  myoglobulin  and  myoalbumin,  which  exist  to  a  very  slight  extent  and 
are  perhaps  only  derived  from  the  remaining  lymph,  belong  to  the  first 
group,  and  the  stroma  substances  of  the  muscle -tubes  belong  to  the  second 
group. 

Myosin  was  first  discovered  by  Kuhxe,  and  constitutes  the  principal 
mass  of  the  soluble  proteins  of  the  dead  muscle.  It  is  generally  considered 
as  the  most  essential  coagulation  product  of  muscle-plasma.  The  name 
myosin  Kuhxe  also  gives  to  the  mother-substance  of  the  plasma-clot,  and 
this  mother-substance  foi-ms,  according  to  certain  investigators,  the  chief 
mass  of  contractile  protoplasm.  The  statements  as  to  the  occurrence  of 
myosin  in  other  organs  besides  the  muscles  require  further  proof.  The 
quantity  of  myosin  in  the  muscles  of  different  animals  varies,  according 
to  Danilewsky,!  between  30  and  110  p.  m. 

Myosin,  as  obtained  from  dead  muscles,  is  a  globulin  whose  elementan,' 
composition,  according  to  Chittexdex  and  Cummixs,^  is,  on  an  average, 
the  following:  C  52.28,  H  7.11,  N  16.77,  S  1.27,  O  22.03  per  cent.      If  the 


'  Zeitschr.  f.  physiol.  Chem.,  7. 

^  Studies  from  the  Physiol.  Chem.  Laboratory  of  Yale  College,  New  Haven,  3,  115. 


450  MUSCLES. 

>jXiLyosin  separates  as  fibres,  or  if  a  myosin  solution  with  a  minimum  quantity 
of  alkali  is  allowed  to  evaporate  on  a  microscope-slide  to  a  gelatinous  mass, 
doubly  refracting  myosin  may  be  obtained.  Myosin  has  the  general  prop- 
erties of  the  globulins.  It  is  insoluble  in  water,  but  soluble  in  dilute  saline 
solutions  as  well  as  in  dilute  acids  or  alkalies,  which  readily  convert  it  into 
albuminates.  It  is  completely  precipitated  upon  saturation  with  NaCl,  also 
by  MgS04,  in  a  solution  containing  94  per  cent  of  the  salt  with  its  water  of 
crystallization  (Halliburton).  The  precipitated  myosin  becomes  insoluble 
readily.  Like  fibrinogen  it  coagulates  at  56°  C.  in  a  solution  containing 
common  salt,  but  differs  from  it  since  under  no  circumstances  can  it  be 
converted  into  fibrin.  The  coagulation  temperature,  according  to  Chitten- 
den and  Cummins,  not  only  varies  for  myosins  of  different  origin,  but  also 
for  the  same  myosin  in  different  salt  solutions. 

Myosin  may  be  prepared  in  the  following  way,  as  suggested  by  Halli- 
burton: The  muscle  is  first  extracted  by  a  5  per  cent  magnesium-sulphate 
solution.  The  filtered  extract  is  then  treated  with  magnesium  sulphate  in 
substance  until  100  c.c.  of  the  liquid  contains  about  50  grams  of  the  salt. 
The  so-called  paramyosinogen  or  musculin  separates.  The  filtered  liquid 
is  then  treated  with  magnesium  sulphate  until  each  100  c.c,  of  the  liquid 
holds  94  grams  of  the  salt  in  solution.  The  myosin  which  now  separates 
is  filtered  off,  dissolved  in  water  by  aid  of  the  retained  salt,  precipitated 
by  diluting  with  water,  and,  when  necessary,  purified  by  redissolving  in 
dilute  salt  solution  and  precipitating  with  water. 

The  older  and  perhaps  the  usual  method  of  preparation  consists,  accord- 
ing to  Danilewsky,^  in  extracting  the  muscle  with  a  5-10  pf:>r  cent  ammo- 
nium-chloride solution,  precipitating  the  myosin  from  the  filtrate  by 
strongly  diluting  with  water,  ard  redissolving  the  precipitate  in  ammonium- 
chloride  solution,  and  the  myosin  obtained  from  this  solution  is  repre- 
cipitated  either  by  diluting  with  water  or  by  removing  the  salt  by  dialysis. 

Musculin,2  called  paramyosinogen  by  Halliburton,  and  myosin 
by  V.  FiJRTH,  is  a  globulin  which  is  characterized  by  its  low  coagulation 
temperature,  about  47°  C,  which  may  vary  in  different  species  of  animals 
(45°  in  frogs,  51°  C.  in  birds).  It  is  more  easily  precipitated  than  myosin 
by  NaCl  or  MgS04  (50  per  cent  salt,  including;  water  of  crystallization). 
According  to  v.  FDrth  it  is  precipitated  by  ammonium  sulphate  with  a 
concentration  of  12-24  per  cent  salt.  If  the  dead  muscle  is  extracted  with 
water  a  part  of  the  musculin  goes  into  solution  and  may  be  precipitated 
therefrom  by  carefully  acidifying.  It  separates  from  a  dilute  salt  solution 
on  dialysis.     Musculin  readily  passes  into  an  insoluble  modification  which 

*  Zeitschr.  f.  physiol.  Chem.,  5,  158. 

2  As  we  have  up  to  the  present  no  conclusive  basis  for  the  identity  of  the  globulins 
called  myosin  and  paramyosinogen,  and  also  as  the  use  of  the  name  myosin  for  the  last- 
mentioned  substance  may  readily  cause  confusion,  the  author  does  not  feel  justified 
in  dropping  the  old  name  musculin  (Nasse). 


PROTEINS   OF  THE   MUSCLE.  451 

V.  FuRTH  calls  myosin  fibrin.  Musculin  is  called  myosin  by  v.  Furth,  as 
he  considers  it  nothing  but  myosin.  As  musculin  has  a  lower  coagulation 
temperature  and  has  other  precipitating  properties  for  neutral  salts  than 
the  older  substance  called  myosin,  it  is  difficult  to  concede  to  this  view. 

Myoglohulin.  After  the  separation  of  the  musculin  and  the  myosin  from  the 
salt  extract  of  the  muscle  by  means  of  MgS04  the  myoglobulin  may  be  precipitated 
by  saturating  the  filtrate  with  the  salt.  It  is  similar  to  serglobulin,  but  coagu- 
lates at  G3°  C.  (Halliburton).  Myoalhumin,  or  muscle-albumin,  seems  to  be 
identical  \\'ith  seralbumin  (seralbumin  a,  according  to  Halliburton),  and  prob- 
ably originates  only  from  the  blood  or  the  lymph.  Proteoses  and  peptones  do 
not  seem  to  exist  in  the  fresh  muscles 

After  the  complete  removal  from  the  muscle  of  aJl  protein  bodies  which 
are  soluble  in  water  and  ammonium  chloride,  an  insoluble  protein  remains 
which  only  swells  in  ammonium-chloride  solution,  and  which  forms  with 
the  other  insoluble  constituents  of  the  muscular  fibre  the  "  muscle-stroma." 
According  to  D.ixilewsky  the  amount  of  such  stroma  substance  is  con- 
nected with  the  muscle  activity.  He  maintains  that  the  muscles  contain 
a  greater  amount  of  this  substance,  compared  with  the  myosin  present, 
when  the  muscles  are  quickly  contracted  and  relaxed. 

According  to  J.  Holmgren. ^  this  stroma  substance  does  not  belong  to 
either  the  nucleoalbumin  or  the  nucleoproteid  group.  It  is  not  a  gluco- 
proteid,  as  it  does  not  yield  a  reducing  substance  when  boiled  with  dilute 
mineral  acids.  It  is  very  similar  to  the  coagulable  proteins  and  dissolves 
in  dilute  alkalies,  forming  an  albuminate.  The  elementary  composition  of 
this  substance  is  nearly  the  same  as  that  of  myosin.  There  is  no  doubt 
that  the  insoluble  substances,  myofibrin  and  myosin  fibrin,  which  are 
formed,  according  to  v.  Furth,  in  the  coagulation  of  the  plasma,  occur  also 
among  the  stroma  substances.  When  the  muscles  are  previously  extracted 
with  water  the  stroma  substance  also  contains  a  part  of  the  myosin  hereby 
made  insoluble.  To  the  proteins  insoluble  in  water  and  neutral  salts 
belongs  the  nucleoproteid  detected  by  Pekelharing,  which  occurs  as 
traces  and  is  soluble  in  faintly  alkaline  water,  and  which  originates  probably 
from  the  muscle  nuclei.  According  to  Bottazzi  and  Ducceschi  -  the  heart 
muscle  is  richer  in  nucleoproteid  than  the  skeletal  muscle. 

Muscle-syntonin,  which  may  be  obtained  by  extracting  the  muscles  with 
hydrochloric  acid  of  1  p.  m.,  and  which,  according  to  K.  Morner,  is  less  -oluble 
and  has  a  greater  aptitude  to  precipitate  than  other  acid  albumins,  seems 
not  to  occur  preformed  in  the  muscles.  Heubner's  ^  mytolin  is  modified  muscle- 
proteid,  chiefly  myosin,  which  has  lost  a  part  of  its  sulphur  by  the  action  of 
alkali. 


'  See  foot-note  1,  p.  44S. 

2  Pekelharing,  Zeitschr.  f.  physiol.  Chem.,  22;    Bottazzi  and  Ducceschi,  Centralbl 
f.  Physiol.,  12. 

'  Arch.  f.  exp.  Pathol,  u.  Pharm.,  iiS. 


452  MUSCLES. 

Proteins  of  the  Muscle-plasma.  As  above  stated,  myosin  was  ordinarily 
considered  as  the  coagulated  modification  of  a  soluble  protein  existing  in  the 
muscle-plasma.  As  in  blood-plasma  there  is  present  a  mother-substance 
of  fibrin,  fibrinogen,  so  also  there  exists  in  the  muscle-plasma  a  mother- 
.substance  of  myosin,  a  soluble  myosin  or  a  myosinogen.  This  body  has 
not  thus  far  been  isolated  with  certainty.  Halliburton,  who  has  detected 
in  the  muscles  an  enzyme-like  substance,  "myosin  fer77ient,"  which  is  related 
"to  fibrin  ferment  but  not  identical  with  it,  has  also  found  that  a  solution 
of  purified  myosin,  in  dilute  salt  solution  (5  per  cent  MgS04),  and  suffi- 
ciently diluted  with  water,  coagulates  after  a  certain  time,  and  at  the 
same  time  becomes  acid,  and  a  typical  myosin-clot  separates.  This  coagu- 
lation, which  is  accelerated  by  warming  or  by  the  addition  of  myosin  fer- 
ment, is,  according  to  Halliburton,  a  process  analogous  to  the  coagulation 
of  the  muscle-plasma.  According  to  this  same  investigator,  myosin  when 
dissolved  in  water  by  the  aid  of  a  neutral  salt  is  reconverted  into  myosino- 
gen, while  after  diluting  with  water  myosin  is  again  produced  from  the 
myosinogen.  The  musculin  (paramyosinogen)  is  carried  down,  according 
to  Halliburton,  with  the  myosin-clot,  but  has  nothing  to  do  with  the 
coagulation,  as  the  myosin-clot  forms  also  in  the  absence  of  musculin,  and 
this  last  is  not  changed  into  myosin. 

Besides  the  traces  of  globulin  and  albumin,  which  perhaps  do  not  belong 
to  the  muscle-plasma,  there  occur  in  mammals,  according  to  v.  Furth,  two 
proteins,  namely,  musculin  (myosin  according  to  v.  FDrth)  and  myogen. 

Musculin  (Nasse)  =  paramyosinogen  (Halliburton)  =  myosin  (v. 
FtJRTH)  forms  about  20  per  cent  of  the  total  proteins  of  the  muscle-plasma 
of  rabbits.  Its  properties  have  already  been  given,  and  it  is  sufficient  to 
remark  that  its  solutions  become  cloudy  on  standing,  and  a  precipitate  of 
myosin  fibrin  occurs,  which  is  insoluble  in  salt  solutions. 

Myogen,  or  myosinogen  (Halliburton),  forms  the  chief  mass,  75-80 
par  cent,  of  the  proteins  of  rabbit  muscle-plasma.  It  does  not  separate 
from  its  solutions  on  dialysis  and  is  not  a  true  globulin,  but  a  protein  sui 
generis.  It  coagulates  at  55-65°  C.  and  is  precipitated  in  the  presence  of 
26-40  per  cent  ammonium  sulphate.  Myogen  solutions  are  precipitated 
by  acetic  acid  only  in  the  presence  of  some  salt.  It  is  converted  into  an 
albuminate  by  alkalies,  this  albuminate  being  precipitable  by  ammonium 
chloride.  ]\Iyogen  passes  spontaneously,  especially  with  higher  tempera- 
tures as  well  as  in  the  presence  of  salt,  into  an  insoluble  modification, 
myogen  fibrin.  A  protein,  coagulating  at  30-40°  C,  soluble  myogen  fibrin, 
is  produced  as  a  soluble  intermediate  step.  This  substance  occurs  to  a 
considerable  extent  in  native  frog-muscle  plasma.  It  does  not  always 
occur  in  the  muscle-plasma  of  warm-blooded  animals,  and  when  it  does 
it  is  present  only  to  a  slight  extent.  It  can  be  separated  by  precipitating 
with  salt  or  by  diffusion.    Halliburton's  assumption  as  to  the  action  of 


PROTEINS   OF  THE    MUSCLE  PLASMA.  453 

a  special  myosin  ferment  has  not  sufficient  basis,  according  to  v.  Furth, 
nor  has  the  often-admitted  analogy  with  the  coagulation  of  the  blood. 
The  difference  between  the  musculin  and  the  myogen  in  their  becoming 
insoluble  is  that  the  musculin  passeg  into  myosin  fibrin  without  any  sol- 
uble intermediate  steps. 

Myogen  may  be  prepared,  according  to  v.  Furth,  by  heating  for  a  short 
time  the  dialyzed  and  filtered  plasma  to  52°  C,  separating  it  in  this  way  from 
the  rest  of  the  musculin.  The  myogen  exists  in  the  new  filtrate  and  can  be 
precipitated  by  ammonium  sulphate.  The  musculin  may  also  be  removed 
by  adding  28  per  cent  ammonium  sulphate  and  then  precipitating  the 
myogen  from  the  filtrate  by  saturating  with  the  salt. 

Stewart  and  Sollmanx  admit  of  only  two  soluble  proteins  in  the 
muscles.  One  is  the  paramyosinogen,  which  is  the  same  as  v.  Furth's 
myosin +the  soluble  myogen  fibrin.  The  other  they  call  myosinogen, 
which  corresponds  to  v.  Furth's  myogen  or  to  Halliburton's  myosinogen 
+  myoglobulin.  It  is  an  atypical  globulin  which  coagulates  at  50-60°  C. 
The  paramyosinogen  as  well  as  the  myosinogen  are  readily  converted 
into  an  insoluble  modification,  myosin.  The  myosin  of  the  above  investi- 
gators is  the  same  as  v.  Furth's  myosin  filjrin  +myogen  fibrin,  and  cor- 
responds, it  seems,  also  to  myosin  mixed  with  paramyosinogen  (Halli- 
burton). Stewart  and  Sollmann  differ  from  Halliburton  in  con- 
sidering that  paramyosinogen  al^o  coagulates  and  is  converted  into  myosin. 
According  to  them  myosin  is  also  insoluble  in  a  NaCl  solution. 

The  views  of  the  various  investigators  differ  so  essentially  and  the 
nomenclature  is  so  complicated  (four  different  things  are  designated  by 
the  name  myosin)  that  it  is  extremely  difficult  to  give  any  correct  review 
of  the  various  notions.^  Thorough  investigations  on  this  subject  are  very 
necessary. 

Myoproteid  is  a  proteid  found  by  v.  Fijrth  in  the  plasma  from  fish- 
muscles.  It  does  not  coagulate  on  boiling,  is  precipitated  by  acetic  acid, 
and  considered  as  a  compound  proteid  by  v.  Furth. 

In  connection  with  v.  Furth's  work,  Przibram  has  carried  on  investigations 
on  the  occurrence  of  muscle-proteins  in  various  classes  of  animals.  The  myosin 
(v.  Furth)  and  myogen  occur  in  all  classes  of  vertebrates;  the  myogen  is  always 
absent  in  the  invertebrates.  Myoproteid  occurs,  at  least  in  considerable  quantity, 
only  in  fishes.  In  the  muscle  after  cutting  the  nerve,  Steyrer'  found  somewhat 
more  musculin  and  less  myogen  in  the  muscle-juice  than  in  the  normal  muscle. 

Muscle-jrigments.  There  is  no  question  that  the  red  color  of  the  muscles, 
even  when  completely  freed  from  blood  depends  in  part   on  haemoglobin. 

'  For  these  reasons  the  author  is  not  sure  whether  he  has  understood  and  correctly 
pven  the  work  of  the  different  investigators. 

*  Przibram,  Hofmeister's  Beitrage,  2;    Steyrer,  ibid.,  4. 


454  MUSCLES. 

K.  MoRNER  has  sho-v\Ti  that  muscle-haemoglobin  is  not  quite  identical  with 
blood-hgemoglobin.  The  statement  of  Mac^Iuxn,  that  in  the  muscles 
another  pigment  occurs  which  is  allied  to  haemochromogen  and  called  myo- 
hcematin  by  him,  has  not  been  substantiated,  at  least  for  muscles  of  higher 
animals  (Levy  and  Morner  i).  MacMuxx  claims  that  myohaematin 
occurs  in  the  muscles  of  insects,  which  do  not  contain  any  haemoglobin. 
The  reddish-yellow  coloring-matter  of  the  muscles  of  the  salmon  has  been 
little  studied. 

Various  enzymes  have  been  found  in  the  muscles.  To  these  belong 
(besides  traces  of  fibrin  ferment  and  myosin  ferment)  the  catalases  and 
oxidases,  which  occur  only  to  a  slight  extent.  The  disputed  glycolytic 
enzyme  (Chapter  VIII),  whose  nature  is  unknown,  probably  belongs  to 
the  oxidases.  An  amylolytic  and  a  proteolytic  enzyme  (Hedix  and  Row- 
LAXD  2)  have  also  been  found,  and  the  hydrol}'tic  and  oxidizing  enzymes 
(Chapter  XV)  active  in  the  formation  and  destruction  of  uric  acid  are  also 
present. 

Extractive  Bodies  of  the  Muscles. 

The  nitrogenous  extractives  consist  chiefly  of  creatine,  on  an  average  of 
1-4  p.  m.  in  the  fresh  muscles  containing  water,  also  the  purine  bases, 
hypoxanthine  and  xanthine,  besides  guanine  and  carnine,  but  chiefly  hypo- 
xanthine.  The  purine  bases  probably  do  not  occur  as  such  but  as  complex 
combinations.  The  quantity  of  nitrogen  as  purine  bases  amounts,  accord- 
ing to  BuRiAX  and  Hall,  in  the  fresh  flesh  of  the  horse,  ox,  and  calf  to 
0.55,  0.63,  and  0.71  p.  m.  respectively,  or  1.3-1.7  p.  m.,  calculated  as  hypo- 
xanthine. In  the  embryonic  ox-muscles,  Kossel  ^  found  more  guanine 
than  hypoxanthine.  The  purine  bases  are  produced  in  the  muscles  them- 
selves, and  their  production,  which  also  takes  place  while  at  rest,  is  greatly 
increased  during  work  (Burian^). 

Among,  the  apparently  habitually  occurring  nitrogeneous  extractives, 
we  should  also  mention  phosphocarnic  acid  as  well  as  inosinic  acid,  which 
is  perhaps  allied  to  it,  carnosinc,  carnitine,  and  perhaps  also  other  bodies 
which  have  recently  been  found  in  meat  extract  and  which  will  be  mem- 
tioned  later. 

Among  the  extractive  substances  is  also  found  the  acid  noticed  by  Limpricht 
in  the  flesh  of  certain  cyprinidea,  namely,  the  nitrogenized  protic  acid,  while  the 
isocreatinine  found  by  J.  Thesen  in  fish-flesh  is  nothing  but  impure  creatinine, 

*  See  MacMunn,  Phil.  Trans,  of  Roy.  Soc,  177,  part  1,  Journ.  of  Physiol.,  8,  and 
Zeitschr.  f.  physiol.  Chem.,  13;  Le\^,  ibid.,  13;  K.  Morner,  Nord.  Med.  Archiv,  Fest- 
band,  1897,  and  Maly's  Jahresber.,  27. 

*  Zeitschr.  f.  physiol.  Chem.,  32. 

^  Burian  and  Hall,  Zeitschr.  f.  physiol.  Chem.,  38;  Kossel,  ibid.,  8,  408. 
*Ibid.,  43. 


CREATINE.  455 

according  to  Poulsson,  Schmidt  and  Korndorfer.'  Uric  acid,  urea,  tavrine, 
and  leucine  are  found  as  traces  in  the  muscles,  in  certain  cases  only  in  a  few  species 
of  animals.  In  regard  to  the  amounts  of  these  different  extractives  in  the  muscles, 
Krukenberg  and  Wagner  ^  have  shown  that  it  varies  greatly  in  different 
animals  A  large  quantity  of  urea  is  found  in  the  muscles  of  the  shark  and  ray; 
uric  acid  is  found  in  alligators;  taurine  in  cephalopoda;  glycocoll  in  gasteropoda, 
and  creatinine  especially  in  fishes.  Ihe  reports  are  very  contradictory  in  regard 
to  the  occurrence  of  ui'ea  in  the  muscles  of  higher  animals.  According  to  the 
investigations  of  Kaufmann  and  Schondurff,  confirmed  by  Brunton-Blaikie,^ 
urea  is  a  regular  constituent  of  the  muscles,  although  M.  Nencki  and  Kowarsk] 
dispute  this. 

The  xanthine  bodies,  with  the  exception  of  carnine,  have  been  treated 
on  pages  159-162,  and  therefore  among  the  extractive  bodies  we  will  first 
consider  the  creatine. 

/NH2 

Creatine,  C4H9N3O2,  (HN)C<  ,  or  methylguanidine- 

\N(CH3).CH2.COOH 
acetic  acid,  occurs  in  the  muscles  of  vertebrate  animals  in  variable  amounts 
in  different  species;  the  largest  quantity  is  found  in  birds.  It  is  also  found 
in  the  brain,  blood,  transudates,  amniotic  fluid,  and  sometimes  also  in  the 
urine.  Creatine  may  be  prepared  synthetically  from  cyanamide  and 
sarcosine  (methylglycocoU).  On  boiling  vnth  baryta  water  it  decomposes 
^with  the  addition  of  water  and  yields  urea,  sarcosine,  and  certain  other 
products.  Because  of  this  behavior  several  investigators  consider  creatine 
as  a  step  in  the  formation  of  urea  in  the  organism.  On  boiling  with  acids, 
creatine  is  easily  converted,  with  the  elimination  of  water  into  creatinine, 
C4H7N3O,  which  occurs  in  urine,  and  which  has  also  been  found  in  the 
muscles  of  the  dog  by  Monari  ^  (see  Chapter  XV),  and  probably  is  a  regular 
constituent  of  the  muscles. 

Creatine  cn,'stallizes  in  hard,  colorless,  monoclinic  prisms  which  lose 
their  water  of  crv^stallization  at  100°  C.  It  i=;  soluble  in  74  parts  of  water 
at  the  ordinary  temperature  and  9419  parts  absolute  alcohol.  It  dissolves 
more  easily  with  the  aid  of  heat.  Its  waterj^  solution  has  a  neutral  reaction. 
Creatine  is  not  dissolved  by  ether.  If  a  creatine  solution  is  boiled  with 
precipitated  mercuric  oxide,  this  is  reduced,  especially  in  the  presence  of 
alkali,  to  mercury  and  oxalic  acid,  and  the  foul-smelling  methyluramine 
(methylguanidine)  is  developed.     A  solution  of  creatine  in  water  is  not  prc- 


*  See  Limprlcht,  Amial.  d.  Chem.  u.  Pharm.,  127,  and  Thesen,  Zeitschr.  f.  physiol, 
Chem.,  24;  Poulsson,  Arch.  f.  exp.  Path.  u.  Pharm.,  51;  Schmidt  and  Korndorfer. 
ihid.,  51. 

2  Zeitschr.  f.  Biologie,  21;  see  also  M.  Henze,  Zeitschr.  f.  physiol.  Chem.,  43; 
Mendel,  Hofmeister's  Beitrage.  5;  Kelly,  ibid..  5. 

^Kaufmann,  Arch,  de  Physiol.  (.5),  6;  Schbndorff,  Pfliiger's  Arch.,  62;  Nencki 
and  Kowarski,  Arch.  f.  exp.  Path.  u.  Pharm.,  36;  Brimton-Blaikie,  Journ.  of  Physiol., 
123.  Supplement. 

*Maly's  Jahresber.,  19,  296. 


456  MUSCLES 

cipitated  by  basic  lead  acetate,  but  gives  a  white,  flaky  precipitate  with 
mercurous  nitrate  if  the  acid  reaction  is  neutraUzed.  ^'\llen  boiled  for  an 
hour  with  dilute  hydrochloric  acid  creatine  is  converted  into  creatinine  and 
may  be  identified  by  its  reactions.  On  boiling  with  formaldehyde  it  can 
be  transformed  into  dioxymethylenecreatinine,  which  crystalUzes  readily 
(Jaffe  1). 

The  preparation  and  detection  of  creatine  is  best  performed  by  the 
following  method  of  Neubauer,^  which  was  first  used  in  the  preparation 
of  creatine  from  muscles:  Finely  cut  meat  is  extracted  with  an  equal  \\ eight 
of  water  at  50°  to  55°  C.  for  10-15  minutes,  pressed,  and  extracted  again 
with  water.  The  proteins  are  removed  from  the  united  extracts  as  far  as 
possible  by  coagulation  at  boiling  heat,  the  filtrate  precipitated  by  the  care- 
ful addition  of  basic  lead  acetate,  the  lead  removed  from  this  filtrate  by  H2S 
and  the  solution  then  carefully  concentrated  to  a  small  volume.  The 
creatine,  which  crystalUzes  in  a  few  days,  is  collected  on  a  filter,  washed 
with  alcohol  of  88  per  cent,  and  purified,  when  necessary,  by  recrystalliza- 
tion.  The  quantitative  estimation  of  creatine  is  performed  according  to  the 
same  method. 

Gamine,  C6H8N4O3+H2O,  is  one  of  the  substances  found  by  Weidel  in 
American  meat  extract.  It  has  also  been  found  by  Krukenberg  and 
Wagner  in  frog-muscles  and  in  the  flesh  of  fishes,  and  by  Pouchet  ^  in  the 
urine.     Carnine  may  be  transformsd  into  hypoxanthine  by  oxidation. 

Carnine  has  been  obtained  as  a  white  crystalline  mass.  It  dissolves 
with  difficulty  in  cold  water,  but  more  readily  in  warm.  It  is  insoluble  in 
alcohol  and  ether.  It  dissolves  in  warm  hydrochloric  acid  and  yields  a  salt 
crystallizing  in  shining  needles,  which  gives  a  double  compound  with 
platinum  chloride.  Its  watery  solution  is  precipitated  by  silver  nitrate* 
but  this  precipitate  is  dissolved  neither  by  ammonia  nor  by  warm  nitric 
acid.  Carnine  does  not  give  the  so-called  Weidel's  xanthine  reaction. 
Its  watery  solution  is  precipitated  by  basic  lead  acetate;  but  the  lead 
compound  may  be  dissolved  on  boiling. 

Carnine  is  prepared  1  y  the  follouing  method:  The  meat  extract  diluted 
with  water  is  completely  precipitated  by  baryta-water.  The  filtrate  is 
precipitated  by  basic  lead  acetate,  the  lead  precipitate  boiled  with  water, 
filtered  while  hot,  and  sulphuretted  hydrogen  passed  through  the  filtrate. 
Remove  the  lead  sulphide  from  the  filtrate  and  concentrate  strongly.  The 
concentrated  solution  is  now  completely  precipitated  with  silver  nitrate, 
the  precipitate  washed  free  from  silver  chloride  by  ammonia,  and  the  carnine 
silver  oxide  suspended  in  water  and  treated  with  sulphuretted  hydrogen. 


*  Ber.  d.  d.  chem.  Gesellsch.,  35. 

^Zeitschr.  f.  physiol.  Chem.,  2  and  6. 

'Weidel,  Annal.  d.  Chem.  u.  Pharm.,  158;  Krukenberg  and  Wagner,  Sitzungsber, 
d.  Wiirzb.  phys.-med.  Gesellsch.,  1883;  Pouchet,  cited  from  Neubauer-Huppert, 
Analyse  des  Harnes,  10.  Aufl.,  335. 


BASES  AND  PHOSPHOaiRXIC  ACID.  457 

Camosine,  CgHj^X^Oj,  has  been  isolated  by  GuLET^^TSCH  and  Amir.\zdibi  ^ 
from  meat  extracts.  It  is  a  base  which  is  perhaps  related  to  arginine,  and  is 
readily  soluble  in  water,  crystallizing  in  flat  needles.  It  is  precipitated  by 
phosphotungstic  acid  and  by  silver  nitrate  in  the  presence  of  an  excess  of  barium 
hydrate  and  forms  a  copper  compound  which  crystallizes  in   hexagonal  plates. 

Carnitine,  €71155X03  (?),  is  another  base  isolated  by  GuLE■^^^TSCH  and  Krimberg  ^ 
from  meat  extracts,  has  a  strong  alkaline  reaction,  and  is  very  readily  soluble 
in  water.  It  gives  a  crystalline  chloroplatinate,  as  well  as  salts  with'HCl  and 
HXO3  which  are  very  readily  soluble  in  water.  The  HXO3  salt,  which  is  also 
crystalline,  is  strongly  levorotatory. 

From  LiEBiG's  extract  of  beef  Kutscher  has  recently  isolated  a  series  of 
new  bodies,  namely,  ignotine,  CyHj^  y^ fi^jCarnomuscarine,  neosmrjCoHnXU^,  novaine, 
C7H17XO2,  viethylguanidine  (also  found  by  Gulewitsch),  and  a  crvstallizable 
chloroplatinate,  Ci8HasX„05.2HCl.PtCI„  of  a  body  which  he  calls  ohUtine.  Zuxz  ^ 
has  also  been  able  to  isolate  from  fresh  muscles  the  three  hexone  bases,  leucine, 
aspartic  acid,  and  glutamic  acid.  He  has  not  decided  whether  these  bodies  exist 
preformed  in  the  muscles. 

The  base  musculamine,  isolated  by  Etard  and  Vila  on  the  hydrolysis  of  veal, 
is  nothing  but  cadaverine,  according  to  Posternak.* 

Inosinic  acid  has  been  discussed  on  page  155.  We  must  also  include  among 
the  nitrogenous  extractives  those  bodies  which  were  first  discovered  by  Gautier  ^ 
and  which  occur  only  in  very  small  cjuantities,  namely,  the  leucomaines,  xantho- 
creatinine,  CJiioSfi,  crusocreatinine,  CjHgX^O,  arnphicrcatine,  C9H19X7O4,  and 
psetidoxantMne,  C4H.X5O. 

In  the  analysis  of  meat  and  for  the  detection  and  separation  of  the  various 
extractive  bodies  of  the  same  we  make  use  of  the  systematic  method  as  suggested 
by  Gautier,'  for  details  of  which  the  reader  is  referred  to  the  original  article. 

Phosphocamic  acid  '  is  a  complicated  substance,  first  isolated  by  Siegfried 
from  meat  extracts,  which  yields  as  cleavage  products  succinic  acid,  paralactic 
acid,  carbon  dioxide,  phosphoric  acid,  and  a  carbohydrate  group,  besides  the 
previously  mentioned  carnic  acid,  which  is  identical  with  or  nearly  related  to 
antipeptone.  It  stands,  according  to  Siegfried,  in  close  relationship  to  the 
nucleins,  and  as  it  yields  peptone  (carnic  acid),  it  is  designated  as  a  nucleon  by 
Siegfried.  Phosphocamic  acid  may  be  precipitated  as  an  iron  compound, 
carnijcrrine,  from  the  extract  of  the  muscles  free  from  proteins.  The  quantity  of 
phosphocamic  acid,  calculated  as  carnic  acid,  can  be  determined  by  multipl^-ing 
the  quantity  of  nitrogen  in  the  compound  by  the  factor  6.1237  (Balke  and 
Ide).  In  this  way  Siegfried  found  0.57-2.4  p.  m.  carnic  acid  in  the  resting 
muscles  of  the  dog,  and  M.  Mijller  1-2  p.  m.  in  the  muscles  of  adults  and  a  maxi- 
mum of  0.57  p.  m.  in  those  of  new-born  infants.  According  to  Cavazzaxi  nucleon 
occurs  to  a  much  greater  extent  in  oysters,  namely,  an  average  of  3.725  p.  m.  It 
also  occurs,  as  he  and  Manicardi  found,  in  the  plant  kingdom.  Phosphocamic 
acid  has  not  been  prepared  in  the  pure  state  and  possesses   on  this  account  a 

•  Zeitschr.  f.  physiol.  Chem.,  30. 

^  Kutscher,  Zeitschr.  f.  Unters.  d.  Nahrungs-  u.  Genussmittel,  10,  and  Centralbl.  f. 
Physiol.,  19;  Zunz,  reference,  ibid.,  18;  Gulewitsch,  Zeitschr.  f.  physiol.  Chem.,  47. 

*  Etard  and  Vila,  Compt.  rend.,  135;  Postemak,  ibid.,  135. 
5  See  Maly's  Jahresber.,  16,  523. 

'>7imi.,22,  335. 

'  In  regard  to  carnic  acid  and  phosphocamic  acid,  see  the  works  of  Siegfried,  Arch, 
f.  (Anat.  u.)  Physiol.,  1894,  Ber.  d.  deutsch.  chem.  GeseUsch.,  2S,  and  Zeitschr. 
f.  physiol.  Chem.,  21  and  28;  M.  MiJller,  t6tJ.,  22;  Kriiger,  iWrf.,  22  and  28;  Balke  and 
Ide,  ibid.,  21,  and  Balke,  ibid.,  22;  Macleod,  ibid.,  28;  E.  Cavazzani,  Centralbl.  f. 
Physiol.,  18,  666;  Panella,  Maly's  Jahresber.,  34. 


458  MUSCLES. 


variable  composition;  according  to  Siegfried  it  serves  as  a  source  of  energy  in 
the  muscles  and  is  consumed  during  work.  Besides,  by  means  of  its  property 
of  forming  soluble  salts  with  the  alkaline  earths,  as  also  an  iron  combination 
soluble  in  alkalies,  it  acts  as  a  means  of  transportation  for  these  bodies  in  the 
animal  body. 

Phosphocarnic  acid  is  prepared  from  the  extract  free  from  protein  by  first 
removing  the  phosphate  by  CaCL  and  XH^.  The  acid  is  precipitated  as  carnifer- 
rine  by  ferric  chloride  from  the  filtrate  while  boiling. 

The  non-nitrogenous  extractive  bodies  of  the  muscles  are  inosite,  glyco- 
gen, sugar,  and  lactic  acid. 

I-osite,  CeH  206+H20=C6H6(OH)6+H20.  This  body,  discovered  by 
ScHERER,  is  not  a  carbo'aydrate,  but  a  hexahydrox}'benzene  (]\Iaquexxe  ^). 
With  hydriodic  acid  it  yields  benzene  and  tri-iodophenol.  Inosite  is  found 
in  the  muscles,  liver,  spleen,  leucocytes,  kidneys,  suprarenal  capsule,  lungs, 
brain,  testicles,  and  in  the  urine  in  pathological  cases,  and  as  traces  in 
normal  urine.  It  is  found  verj^  widely  distributed  in  the  vegetable  king- 
dom, especially  in  the  unripe  fruit  of  green  beans  (Phaseolus  ^nllgaris),  and 
therefore  it  is  also  called  phaseomaxnite.  According  to  Wixtersteix  a 
phosphorized  compound  occurs  in  the  vegetable  kingdom  which  yields 
inosite  as  a  cleavage  product.  This  compound  is,  according  to  Posterxak ,2 
probably  ox\methylphosphoric  acid,  which  also  yields  inosite  on  decom- 
position by  condensation. 

Inosite  cry^stallizes  in  large,  colorless,  rhomljic  crystals  of  the  monoclinic 
system,  or,  if  not  pure  and  if  only  a  small  quantity  crj'stallizes,  it  forms 
groups  of  fine  cr}^stals  similar  to  cauliflower.  It  loses  its  water  of  crystalliza- 
tion at  110°  C,  also  if  exposed  to  the  air  for  a  long  time.  Such  exposed 
crystals  are  non-transparent  and  milk-white.  The  crj'stals  melt  at  225°  C. 
when  dr}^  Inosite  dissolves  in  7.5  parts  of  water  at  ordinary'  temperature, 
and  the  solution  has  a  sweetish  taste.  It  is  insoluble  in  strong  alcohol  and 
in  ether.  It  dissolves  cupric  hydrate  in  alkaline  solutions,  but  does  not 
reduce  on  boiling.  It  gives  negative  results  with  ]\Ioore's  test  and  with 
Bottger-Almex's  bismuth  test.  It  does  not  ferment  with  beer-yeast, 
but  may  undergo  lactic-  and  butyric-acid  fermentation.  The  la^'tic  acid 
formed  thereby  is  sarcolactic  acid  according  to  Hilger,  and  fermenta- 
tion lactic  acid  according  to  Vohl.^  Inosite  is  oxidized  into  rhodizonic 
acid  by  an  excess  of  nitric  acid,  and  the  following  reactions  depend  upon 
this  behavior: 

If  inosite  is  evaporated  to  dryness  on  platinum-foil  with  nitric  acid  and 
the  residue  treated  with  ammonia  and  a  drop  of  calcium-chloride  solution 
and  carefully  re-evaporated  to   drjTiess,   a  beautiful  rose-red  residue  is 

'  Bull,  de  la  see.  chim.  (2j,  47  and  48;   Compt.  rend.,  104. 

^  W^interstein,  Ber.  d.  d.  chem.  Gesellsch.,  30;    Postemak,  Contribution  a  I'etude 
chim.  de  rassiniilation  chlorophyllienne,  Revue  generale  de  Botanique,  12  (1900). 
^  Hilger,  Aunal.  d.  Chem.  u.  Pharm.,  160;   Vohl,  Ber.  d.  d.  chem.  Gesellsch.,  9. 


INOSITE  AND  GLYCOGEN.  459 

obtained  (Scherer's  inosite  test).  If  we  evaporate  an  inosite  solution  to 
incipient  dryness  and  moisten  the  residue  with  a  little  mercuric  nitrate 
solution,  there  is  obtained  a  yellowish  residue  on  drying,  v.luch  becomes  a 
beautiful  red  on  strongly  heating.  The  coloration  disappears  on  cooling, 
but  it  reappears  on  gently  warming  (Ga:  lois'  inosite  test). 

To  prepare  inosite  from  a  liquid  or  from  a  watery  extract  of  a  tissue, 
the  proteii  s  are  first  removed  by  coagulation  at  boiling  heat.  The  filtrate 
is  precipitated  by  sugar  of  lead,  this  filtrate  boiled  with  basic  lead  acetate 
and  allowed  to  stand  24-48  hours.  The  precipitate  thus  obtained,  which 
contains  all  the  inosite,  is  decomposed  in  water  by  H2S.  The  filtrate  is 
strongly  concentrated,  treated  with  2-4  vols,  hot  alcohol,  and  the  liquid 
removed  as  soon  as  possible  from  the  tough  or  flaky  masses  which  ordinarily 
separate.  If  no  ciystals  separate  from  the  liquid  within  twenty-four  hours, 
then  treat  with  ether  until  the  liquid  has  a  milky  appearance  and  allow  it 
to  stand.  In  the  presence  of  a  sufficient  quantity  of  ether,  crj^stals  of 
inosite  separate  within  twenty-four  hours.  The  crj^stals  thus  obtained, 
as  also  those  which  are  obtained  from  the  alcoholic  solution  directly,  are 
recrystallized  by  redissolving  in  very  little  boiling  water  and  addin  •  2-4 
vols,  of  alcohol. 

Glycogen  is  a  constant  constituent  of  the  living  muscle,  while  it  may  be 
absent  in  the  dead  muscle.  The  quantity  of  glycogen  varies  in  the  different 
muscles  of  the  same  animal.  Bohm  ^  found  10  p.  m.  glycogen  in  the  muscles 
of  cats,  and  moreover  he  found  a  greater  amount  in  the  muscles  of  the 
extremities  than  in  those  of  the  rump.  Sch()xdorff  has  found  a  maxi- 
mum of  37.2  p.  m.  in  the  dog-muscle.  The  statements  as  to  the  quantity 
of  glycogen  in  the  heart  differ  somewhat;  although  the  heart  is  considered 
as  somewhat  poorer  in  glycogen  than  the  other  muscles,  still  this  difference 
is  not  very  great  and  can  be  explained  by  the  ready  disappearance  of 
glycogen  from  the  heart  after  death,  as  well  as  after  starvation  and  after 
strong  work  (Boruttau,  Jensen,  Kisch  2).  Work  and  food  have  a 
great  influence  upon  the  quantity  of  glycogen.  Bohm  found  1-4  p.  m. 
glycogen  in  the  muscles  of  fasting  animals,  and  7-10  p.  m.  after  partaking 
of  food.  As  stated  in  Chapter  VIII,  work,  starvation,  and  lack  of  carbo- 
hydrates in  the  food  cause  the  glycogen  to  disappaar  earlier  from  the  liver 
than  from  the  muscles. 

The  sugar  of  the  muscles,  of  which  only  traces  occur  in  the  living  muscle, 
and  W'hich  is  probably  formed  after  the  death  of  the  muscle  from  the  mus- 
cle-glycogen,  is,  according  to  the  investigations  of  Panormoff,  in  part 
dextrose,  but  consists  chiefly  of  maltose  (Osborne  and  Zobel  ^)  with  some 
dextrin. 

>  Bohm,  Pfliiger's  Arch.,  23,  44;  Schoniorff,  ibid.,  99. 

^  Boruttau,  Zeitschr.  f.  physiol.  Chem.,  18;  Jensen,  iW(/.,  35;  Kisch,  Hofmeister'3 
Beitrage,  8. 

^Panormoff.  Zeitschr.  f.  physiol.  Chem.,  17;  Osborne  and  Zobel,  Journ.  of  Phy- 
siol., 29. 


460  MUSCLES. 

Lactic  Acids.  Of  the  oxypropionic  acids  with  the  formula  CsHeOs 
there  is  one,  ethylene  lactic  acid,  CH2(OH).CH2.COOH,  which  is  not  found 
in  the  animal  body  and  therefore  has  no  physiological  chemical  interest. 

CH3 
Indeed  only  a-oxypropionic  acid  or  ethylidene  lactic  acid,  CH(OH),  of 

COOH 
which  there  are  three  physical  isomeres,  is  of  importance.  These  three 
ethylidene  lactic  acids  are  the  ordinary,  optically  inactive  fermentation 
LACTIC  ACID,  the  dextrorotatory  paralactic  or  s-1rcolactic  acid,  and 
the  LEVOLACTic  ACID  obtained  by  Schardinger  by  the  fermentation  of 
cane-sugar  by  means  of  a  special  bacillus.  This  levolactic  acid,  which 
has  also  been  detected  by  Blachstein  in  the  cul  ure  of  Gaffky's  typhoid 
bacillus  in  a  solution  of  sugar  and  peptone,  and  which  is  formed  by  vari- 
ous vibriones,  need  not  be  described  here.^ 

The  fermentation  lactic  acid,  which  is  formed  from  lactose  by  allow- 
ing milk  to  sour  and  by  the  acid  fermentation  of  other  carbohydrates, 
is  considered  to  exist  in  small  quantities  in  the  muscles  (Heintz),  in  the 
gray  matter  of  the  brain  (Gscheidlen),  and  in  diabetic  urine.  The  occur- 
rence of  fermentation  lactic  acid  in  the  brain  and  other  organs  has  recently 
been  disputed  by  Moriya.2  During  digestion  this  acid  is  also  found  in 
the  contents  of  the  stomach  and  intestine,  and  as  alkali  lactate  in  the  chyle. 
The  paralactic  acid  is,  at  all  events,  the  true  acid  of  meat  extracts,  and  this 
alone  has  been  found  with  certainty  in  dead  muscle.  The  lactic  acid  which 
is  found  in  the  brain,  spleen,  lymphatic  glands,  thymus,  thyroid  gland, 
blood,  bile,  pathological  transudates,  osteomalacious  bones,  in  perspira- 
tion in  puerperal  fever,  in  the  urine  after  fatiguing  marches,  in  acute 
yellow  atrophy  of  the  liver,  in  poisoning  by  phosphorus,  and  especially 
after  extirpation  of  the  liver  seems  always  to  be  paralactic  acid. 

The  origin  of  paralactic  acid  in  the  animal  organism  has  been  sought 
by  several  investigators,  who  took  for  basis  the  researches  of  Gaglio, 
Minkowski,  and  Araki,  in  a  decomposition  of  protein  in  the  tissues. 
Gaglio  claims  a  lactic-acid  formation  by  passing  blood  through  the  kid- 
neys and  lungs.  He  also  found  0.3-0.5  p.  m.  lactic  acid  in  the  blood  of 
a  dog  after  protein  food,  and  only  0.17-0.21  p.  m.  after  fasting  for  forty- 
eight  hours.  According  to  Minkowski  the  quantity  of  lactic  acid  elimi- 
nated I  y  the  urine  in  animals  with  extirpated  livers  is  increased  with  pro- 
tein food,  while  the  administration  of  carbohydrates  has  no  effect.  Araki 
has  also  shown  that  if  we  produce  a  scarcity  of  oxygen  in  animals  (dogs, 
rabbits,  and  hens)  by  poisoning  with  carbon  monoxide,  by  the  inhalation 

'See  Schardinger,  Monatshefte  f.  Chem.,  11;  Blachstein,  Arch,  des  sciences  biol. 
de  St.  P^tersbourg,  1,  199;  Kuprianow,  Arch.  f.  Hygiene,  19,  and  Gosio,  ibid.,  21. 

^  Heintz,  Annal.  d.  Chem.  u.  Pharm.,  157,  and  Gscheidlen,  Pfliiger's  Arch.,  8, 
171;   Moriya,  Zeitschrift  f.  physiol.  Chem.,  43. 


LACTIC  ACID.  461 

of  air  deficient  in  oxygen,  or  by  any  other  means,  a  considerable  elimina- 
tion of  lactic  acid  (besides  dextrose  and  also  often  albumin)  takes  place 
through  the  urine,  an  observation  which  has  been  confirmed  by  Saito 
and  KATSUYA^L\.^  As  a  scarcity  of  oxygen,  according  to  the  ordinary' 
statements,  produces  an  increase  of  the  protein  catabolism  in  the  body, 
the  increased  elimination  of  lactic  acid  in  these  cases  must  be  due  in  part 
to  an  increased  proteia  destruction  and  in  part  to  a  diminished  oxidaion. 

Araki  has  not  drawn  such  a  conclusion  from  his  experiments,  but  he 
considers  the  abundant  formation  of  lactic  acid  to  be  due  to  a  cleavage  of 
the  sugar  formed  from  the  glycogen.  He  found  that  in  all  cases  where 
lactic  acid  and  sugar  appeared  in  the  urine  the  quantity  of  glycogen  in 
the  liver  and  muscles  was  always  diminished.  He  also  calls  attention  to 
the  fact  that  dextrolactic  acid  may  be  formed  from  glycogen,  as  directly 
observed  by  Ekuxixa,^  and  also  to  the  numerous  observations  on  the 
formation  of  lactic  acid  and  the  co::sumption  of  glycogen  in  muscular 
activity.  Without  denying  the  possibility  of  a  formation  of  lactic  acid 
from  protein,  he  states  that  with  lack  of  o  :ygen  we  have  to  deal  with  an 
incomplete  combustion  of  the  lactic  acid  derived  by  a  cleavage  of  the  sugar. 
Hoppe-Seyler  3  also  positively  defends  the  view  as  to  the  formation  of 
lactic  acid  from  carbohydrates.  He  was  of  the  view  that  lactic  acid  is 
produced  from  the  carbohydrates  by  the  cleavage  of  the  sugar  only  with 
lack  of  oxygen,  while  with  sufficient  oxygen  the  sugar  is  burned  into  carbon 
dioxide  and  w^ater.  The  formation  of  lactic  acid  in  the  absence  of  free 
oxygen  and  in  the  presence  of  glycogen  or  dextrose  is,  according  to  Hoppe- 
Seyler,  ver}'  probably  a  function  of  all  living  protoplasm.  In  the  anaero- 
bic metabolism  of  the  animal  cells,  according  to  the  recent  investigations 
on  alcoholic  fermentation  in  the  tissues  (see  Chapters  I  and  ^^II),  carbon 
dioxide  and  alcohol  are  formed  from  the  sugar,  with  lactic  acid  as  an  inter- 
mediary step;  but  even  if  this  view  be  correct  and  when  the  cells,  as  Stok- 
LASA^  and  his  collaborators  have  shown,  contain  a  lactic-acid-forming 
enzyme,  it  is  not  known  what  kind  of  lactic  acid  is  here  produced.  Accord- 
ing to  MoRiSHiMA  an  increase  in  the  lactic  acid  in  the  liver  occurs  after 
death,  probably  from  the  liver  glycogen,  but  this  acid  is  chiefly  fermenta- 
tion lactic  acid.  Asher  and  Jackson  °  experimented  by  transfusing  blood 
(with  and  without  the  addition  of  sugar)  through  the  lower  extremities  of 

'  Gaglio,  Arch.  f.  (Anat.  u.)  Physiol.,  1886;  Minkowski.  Arch.  exp.  Path.  u.  Pharm., 
21  and  31;  Araki,  Zeitschr.  f.  physiol.  Chem.,  15, 16,  17,  and  19;  Saito  and  Katsuyama, 
ibid.,  32. 

2  Joum.  f.  prakt.  Chem.  (N.  F.),  21. 

^  Virchow's  Festschrift,  also  Ber.  d.  deut.sch.  chem.  Gesellsch.,  25,  Referatb.,  685. 

*  Simacek,  Centralbl.  T.  Physiol.,  1";  Stoklasa,  Jelinek,  and  Cemy,  ibid.,  16. 

^Morishima,  Arch.  f.  exp.  Path.  u.  Pharm.,  43;  Asher  and  Jackson,  Zeitschr.  f. 
Biologic,  41. 


462  MUSCLES. 

dogs,  and  neither  in  these  experiments  nor  in  those  where  the  larger-organs 
(liver  and  abdominal  \nscera)  were  excluded  from  the  circulation  could 
they  detect  any  increase  of  lactic  acid  due  to  the  sugar. 

Although  these  last-mentioned  investigations  do  not  show  any  forma- 
tion of  lactic  acid  from  carbohydrates,  still,  on  the  other  hand,  we  have 
recent  investigations  that  make  such  an  origin  for  lactic  acid  ver}^  proba- 
ble. Thus  Embden  1  has  found,  on  percolating  blood  through  a  sur\dving 
liver  rich  in  glycogen,  that  lactic  acid  was  formed,  and  also  that  thi^  acid 
was  produced  in  abundance  when  blood  rich  in  sugar  was  transfused  through 
a  glycogen-free  liver,  while,  on  the  contrary,  blood  poor  in  sugar  led  to 
only  a  very  inconsiderable  formation  of  lactic  acid.  The  investigations  of 
A.  R.  Mandel  and  Lusk^  also  indicate  a  formation  of  lactic  acid  from 
carbohydrates  in  the  animal  body.  They  have  shown  that  in  dogs,  after 
phosphorus  poisoning,  an  abundance  of  lactic  acid  occurs  in  the  blood  and 
urine,  and  that  this  disappears  from  these  fluids  on  bringing  about  a  phlor- 
hizm  diabetes  in  the  animal.  Phosphorus  intoxication  caused  no  lactic- 
acid  formation  in  a  phlorhizin- diabetic  dog.  Although  it  is  difficult  to 
give  a  satisfactory  explanation  of  the  results  of  these  experiments,  still  it 
seems  probable  that  by  elimination  of  the  sugar  in  phlorhizin  diabetes  a 
mother-substance  of  the  lactic  acid  is  lost. 

The  carbohydrates,  as  well  as  the  proteins,  it  seems,  must  be  considered 
as  the  material  from  which  the  lactic  acid  is  formed  in  the  body.  In  a 
previous  chapter  (VIII)  we  mentioned  the  fonnation  of  lactic  acid  in  the 
animal  body  by  a  d lamination  of  alanine,  and  this  gives  us  an  indication 
of  a  lactic  acid  formation  from  protein.  Phosphocamic  acid  is  considered 
by  Siegfried  as  another  source  of  sarcolactic  acid. 

The  lactic  acids  are  amorphous.  They  have  the  appearance  of  color- 
less or  faintly  yellowish,  acid-reacting  syrupswhich  mix  in  all  pro  portions 
with  water,  alcohol,  or  ether.  The  salts  are  soluble  in  water,  and  most  of 
them  also  in  alcohol.  The  two  acids  are  differentiated  from  each  other  by 
their  different  optical  properties — paralactic  acid  being  dextrogyrate,  while 
fermentation  lactic  acid  is  optically  inactive — also  by  their  different  solu- 
bilities and  the  different  amounts  of  water  of  crystallization  of  the  calcium 
and  zinc  salts.  The  zinc  salt  of  fermentation  lactic  acid  dissolves  in  58-63 
parts  of  water  at  14-15°  C,  and  contains  18.18  per  cent  water  of  crystalli- 
zation, corresponding  to  the  formula  Zn(C3H503)2  +  3H20.  The  zinc  salt 
of  paralactic  acid  dissolves  in  17.5  parts  of  water  at  the  above  tempera- 
ture and  contains  ordinarily  12.9  per  cent  water,  corresponding  to  the 
formula  Zn(C3H503)2  +  2H20.  The  calcium  salt  of  fermantation  lactic  acid 
dissolves  in  9.5  parts  water  and  contains  29.22  per  cent  (  =  5  molecules) 
water  of  crystallization,  while  calcium  paralactate  dissolves  in  12.4  parts 

»  Centralbl.  f.  PhyaioL,  18,  832.  *  Amer.  Journ.  of  Physiol.,  16. 


LACTIC   ACIDS.  463 

water  and  contains  24.83  or  26.21  per  cent  (  =  4  or  4|  molecules)  water  of 
crystallization.  Both  calcium  salts  crystallize,  not  unlike  tyrosine,  in  spears 
or  tufts  of  very  fine  microscopic  needles.  Hoppe-Seyler  and  Araki^ 
who  have  closely  studied  the  optical  properties  of  the  lactic  acids  and 
lactates,  consider  the  lithium  salt  as  best  suited  for  the  preparation  and 
quantitative  estimation  of  the  lactic  acids.  The  lithium  salt  contains 
7.29  per  cent  Li.  For  further  information  as  to  the  salts  and  specific 
rotation  of  the  lactic  acids  see  Hoppe-Seyler-Thierfelder's  Hand- 
buch,  7.  Aufl.,  1903.1 

Lactic  acids  may  be  detected  in  organs  and  tissues  in  the  following 
manner:  After  complete  extraction  with  water,  the  protein  is  removed  by 
coagulation  at  boiling  temperature  and  the  addition  of  a  small  quantity  of 
sulphuric  acid.  The  liquid  is  then  exactly  neutralized  while  boiling  with 
caustic  baiyta,  and  then  evaporated  to  a  syrup  after  filtration.  The  residue 
is  precipitated  with  absolute  alcohol,  and  the  precipitate  completely  ex- 
tracted with  alcohol.  The  alcohol  is  entirely  distilled  from  the  united  alco- 
holic extracts,  and  the  neutral  residue  is  shaken  with  ether  to  remove  the 
fat.  The  residue  is  dissolved  in  water  and  phosphoric  acid  added,  and  tlie 
solution  repeatedly  shaken  with  fresh  quantities  of  ether,  which  dissolves 
the  lactic  acid.  The  ether  is  now  distilled  from  the  united  ethereal  ex- 
tracts, the  residue  dissolved  in  water,  and  this  solution  carefully  warmed  on 
the  water-bath  to  remove  the  last  traces  of  ether  and  volatile  acids.  A 
solution  of  zinc  lactate  is  prepared  from  this  filtered  solution  by  boiling 
with  zinc  carbonate,  and  this  is  evaporated  until  crj'stallization  commences, 
and  then  is  allowed  to  stand  over  sulphuric  acid.  An  analysis  of  the  salts 
is  necessary  in  careful  work.  According  to  Heffter.^  in  muscles  not  having 
undergone  rigor  mortis  the  lactic  acid  can  be  extracted  more  easily  by  alco- 
hol than  by  water. 

Fat  is  never  absent  in  the  muscles.  Some  fat  is  always  found  in  the 
intermuscular  connective  tissue;  but  the  muscle-fibres  themselves  also 
contain  fat.  The  quantity  of  fat  in  the  real  muscle  substance  is  always 
small,  usually  amounting  to  about  10  p.  m.  or  somewhat  more.  A  con- 
siderable quantit}^  of  fat  in  the  muscle-fibres  is  found  only  in  fatty  degen- 
eration. A  part  of  the  muscle-fat  can  be  readily  extracted,  while  another 
part  can  be  extracted  onl}-  with  the  greatest  difficulty.  This  latter  part, 
it  is  claimed,  exists  finely  divided  in  the  contractile  substance  itself  and  is 
richer  in  free  fatty  acids,  standing,  according  to  Zuntz  and  Bogdanow,^ 
in  close  relationship  to  the  activit}'  of  the  muscles  because  it  is  consumed 
during  work.  LecitJtin  is  a  regular  constituent  of  the  muscles,  and  it  is 
quite  possible  that  the  fat  which  is  difficult  of  extraction  and  which  is  rich 
in  fatty  acids  depends  in  part  on  a  decomposition  of  the  lecithin.     The 

'  See  also  E.  Jungfleisch,  Compt.  rend.,  139,  140,  and  142. 
2  Arch.  f.  exp.  Path.  u.  Phama.,  38. 
5  Arch.  f.  (Anat.  u.)  Physiol.,  1897. 


464  MUSCLES. 

amount  of  lecithin  is  not  considerable.  In  normal  dog-heart,  as  free  from 
fat  as  possible,  Rubo.v  ^  found  that  the  lecithin  amounted  to  7.5-8.5  per 
cent  of  the  drj'  substance;  for  the  striated  muscle  the  amount  of  lecithin 
was  rather  constant,  namely,  5.08  per  cent.  The  ether  extract  of  the  heart 
of  the  dog  contained  60-70  per  cent  lecithin. 

The  Mineral  Bodic:  of  the  Muscles.  The  ash  remaining  after  burning 
the  muscle,  which  amounts  to  about  10-15  p.  m.,  calculated  on  the  moist 
muscle,  is  acid  in  reaction.  The  largest  constituent  of  the  ash  is  potas- 
sium, whose  occurrence,  according  to  Macallum,  is  restricted  to  the  dark 
diagonal  bundles,  and  phosphoric  acid.  Next  in  amount  we  have  sodium 
and  magnesium,  and  lastly  calcium,  chlorine,  and  iron  oxide.  Sulphates 
exist  only  as  traces  in  the  muscles,  but  are  formed  by  the  burning  of  the 
proteins  of  the  muscles,  and  therefore  occur  in  abundant  quantities  in  the 
ash-  The  muscles  contain  such  a  large  quantity  of  potassium  and  phos- 
phoric acid  that  potassium  phosphate  seems  to  be  unquestionably  the  pre- 
dominating salt.  Chlorine  is  found  in  such  insignificant  quantities  that  it 
is  perhaps  derived  from  a  contamination  with  blood  or  lymph.  The  quan- 
tity of  magnesium  is,  as  a  rule,  considerably  greater  than  that  of  calcium. 
Iron  occurs  only  in  very  small  amounts.  Schmey  2  found  variations  be- 
tween 0.0129  p.  m.  (rabbits)  and  0.0793  p.  m.  (liuman),  calculated  on  the 
fresh  muscle  substance.  The  heart-muscle  was  comparatively  richer  in 
iron,  0.06-0.109  p.  m. 

The  importance  of  the  various  mineral  bodies  for  the  function  of  the 
muscjes  has  been  studied  by  several  experimenters  (Loeb,  Lingle,  Howell, 
Overton,  Laxgendorff  and  Hueck,  and  others  ^).  Further  proof  as  to  the 
pre\'iously  discussed  ion  action  of  the  electrolytes  and  the  antagonism  of 
various  ions  has  been  given  by  many  very-  interesting  investigations.  These 
researches  also  indicate  that  each  of  the  ions  Na,  Ca,  and  K  plays  a  certain 
part  in  the  maintenance  of  the  excitability,  in  the  contraction  and  in  the 
fatigue  of  the  muscle  (I'leart);  still  these  investigations  have  not  led  to  con- 
cordant results,  so  that  we  are  not  yet  clear  on  the  action  of  these  ions. 
Nevertheless  it  seems  to  be  established  that  the  combined  action  of  various 
ions  is  a  necessity  for  the  normal  function  of  the  muscles.  It  has  also 
been  shown  that  it  is  possible  to  maintain  the  muscle  (the  heart)  in  regular 
activity  for  a  long  time  by  means  of  a  transfusion  of  liquid  saturated 
with  oxygen  and  which  contained  about  7  p.  m.  NaCl,  besides  small 
amounts  of  CaClg  (0.2  p.  m.),  KCl  (0.1  p.  m.),  and  NaHCOa  (0.1  p.  m.). 


*  Arch.  f.  exp.  Path.  u.  Pharm.,  52. 

2  MacaUum,  Joum.  of  Physiol.,  32;  Schmey,  Zeitschr.  f.  physiol.  Chem.,  39- 

^  Loeb,  Amer.  Joum.  of  Physiol.,  3,  and  Pfliger's  Arch.,  80,  91;    Lingle,  Amer. 

Journ.  of  Physiol.,  4  (also  references  to  literature);   Overton,  Pfluger's  Arch.,  92  and 

105;  Langendorfif  and  Hueck,  ibid.,  96. 


PERMEABILITY   OF  THE    MUSCLES.  465 

The  gases  of  the  muscles  consist  of  large  quantities  of  carbon  dioxide, 
besides  traces  of  nitrogen. 

In  regard  to  the  permeability  of  the  muscles  for  various  bodies  there  are 
the  complete  investigations  of  Overton. ^  The  different  sheaths  of  the 
muscles,  the  sarcolemma  and  perimysium  internum,  offer  no  very  great 
resistance  to  the  diffusion  of  the  most  soluble  crs^stalloid  compounds,  while 
the  muscle-fibres,  on  the  contrarv-  (exclusive  of  the  sarcolemma),  are  almost 
if  not  entirely  impervdous  to  most  inorganic  compounds  and  to  many 
organic  compounds.  The  muscle-fi'jres  themselves  are  actually  semiper- 
meable structures  which  are  permeable  for  water  but  not  for  the  molecules 
or  ions  of  sodium  chloride  and  of  potassium  phosphate.  The  muscle-fibres, 
as  well  as  the  various  sheaths,  are  impermeable  to  colloids. 

The  behavior  of  the  numerous  bodies  investigated  cannot  be  discussed 
in  this  work.  The  general  rule  is  as  follows:  All  compounds  which,  besides 
havirg  a  marked  solubility  in  water,  are  readily  soluble  in  ethyl  ether,  in 
the  higher  alcohols,  in  olive-oil  and  in  similar  organic  solvents,  or  are  not 
much  less  soluble  in  the  last -mentioned  solvents  than  in  water,  pass  through 
the  living  muscle-fibres  with  great  ease.  The  greater  the  difference 
between  the  solubility  of  a  compound  in  water  and  in  the  other  solvents 
mentioned,  the  slower  does  the  passage  into  the  muscle-fibres  take  place. 
The  permeability  changes  essentially  on  the  death  of  the  muscle. 

The  living  muscle-fibres  are  readily  permeable  to  oxygen,  carbon  dioxide, 
and  ammonia,  while  the  hexoses  and  disaccharides  do  not  readily  pass 
into  them.  It  is  very  remarkable  that  a  great  portion  of  those  compounds 
which  take  part  in  the  normal  metabohsm  of  plants  and  animals  belong  to 
those  bodies  to  which  the  muscle-fibres  (and  also  other  cells)  are  entirely  or 
at  least  nearly  impermeable.  On  the  contrar}^,  derivatives  can  be  pre- 
pared from  these  bodies  which  pass  into  the  cells  verj'  readily,  and  Over- 
ton finds  that  it  is  not  impossible  that  the  organism  in  part  makes  use  of 
a  similar  artifice  in  order  to  regulate  the  concentration  of  the  nutritive 
bodies  within  the  protoplasm. 

Rigor  Mortis  of  the  Muscles.  If  the  influence  of  the  circulating  oxA'gen- 
ated  blood  is  removed  from  the  muscles,  as  after  the  death  of  the  animal 
or  by  ligature  of  the  aorta  or  the  muscle-arteries  (Stenson's  test),  rigor 
mortis  sooner  or  later  takes  place.  The  ordinary  rigor  appearing  under 
these  circumstances  is  called  the  spontaneous  or  the  fermentative  rigor, 
because  it  seems  to  depend  in  part  on  the  action  of  an  enzyme.  A  muscle 
may  also  become  stiff  for  other  reasons.  The  muscles  may  become  momen- 
tarily stiff  by  warming,  in  the  case  of  frogs  to  40°,  in  mammalia  to  48-50°, 
and  in  birds  to  53°  C.      The  heat -rigor  depends  upon  the  coagulation  of 

*  Pfliiger's  Arch.,  92.  See  also  Hober,  ibid.,  106,  and  Hamburger,  Osmotischer 
Druck  und  lonenlehre,  Bd.  3. 


466  MUSCLES. 

certain  proteins,  and  its  occurrence  at  lower  temperatures  in  cold-blooded 
as  compared  with  warm-blooded  animals  is  due,  according  to  v.  FtJRTH, 
to  the  fact  that  in  the  first  a  soluble  myogen  fibrin  occurs  preformed  in 
the  muscle  which  coagulates  at  30-40°  C,  while  in  the  warm-blooded 
animals  the  coagulating  substance  is  musculin  (myosin  of  v.  Furth)  which 
coagulates  at  a  higher  temperature.  Distilled  water  may  also  produce 
a  rigor  in  the  muscles  (water-rigor).  Acids,  even  very  weak  ones,  such  as 
carbon  dioxide,  may  quickly  produce  a  rigor  (acid-rigor),  or  hasten  its 
appearance.  A  number  of  chemically  different  substances,  such  as  chloro- 
form, ether,  alcohol,  ethereal  oils,  caffeine,  and  many  alkaloids,  produce  a 
similar  effect.  The  rigor  which  is  produced  by  means  of  acids  or  other 
agents  which,  like  alcohol,  coagulate  proteins  must  be  considered  as  pro- 
duced by  entirely  different  processes  from  those  causing  spontaneous  rigor. 

When  the  muscle  passes  into  rigor  mortis  it  becomes  shorter  and  thicker, 
harder  and  non-transparent,  and  less  ductile.  The  acid  part  of  the  ampho- 
teric reaction  becomes  stronger,  w^hich  is  explained  by  most  investigators 
by  the  assumption  of  a  formation  of  lactic  acid.  There  is  hardly  any  doubt 
that  this  increase  in  acidi;  y  may  at  least  in  part  be  due  to  a  transformation 
of  a  part  of  the  diphosphate  into  monophosphate  by  the  lactic  acid.  The 
statements  in  regard  to  the  presence  or  absence  of  free  lactic  acid  in  the 
rigor-mortis  muscle  are  contradict ory.i  Besides  the  formation  of  acid,  the 
chemical  processes  which  take  place  in  rigor  of  the  muscles  are  the  follow- 
ing: By  the  coagulation  of  the  plasma  a  myosin-clot  is  produced  which  is 
the  cause  of  the  hardening  and  of  the  diminished  transparency  of  the  muscle; 
but  this  view  must  be  changed  on  account  of  the  researches  of  v.  Furth, 
which  have  shown  that  the  clot  consists  of  myogen  fibrin  and  myosin  fibrin. 
The  appearance  of  this  clot  may  be  hastened  by  the  simultaneous  occurrence 
of  lactic  acid.  Carbon  dioxide  is  also  formed,  which  does  not  se3m  to  be 
a  direct  oxidation  product,  but  a  product  of  the  cleavage  processes.  Her- 
mann 2  claims  that  carbon  dioxide  is  produced  in  the  removed  muscle, 
even  in  the  absence  of  oxygen,  when  it  passes  into  rigor  mortis.  In  con- 
nection with  this  view  we  must  call  attention  to  Folin's^  observations 
that  no  protein  coagulation  took  place  in  rigor  under  special  conditions. 

As  many  investigators  admit  of  an  increased  formation  of  lactic  acid  on 
the  appearance  of  rigor  mortis,  the  question  arises,  from  what  constituents 
of  the  muscle  is  this  acid  derived?  The  most  probable  explanation  is  that 
the  lactic  acid  is  produced  from  the  glycogen,  as  certain  investigators,  such 


*  It  is  impossible  to  enter  into  the  details  of  the  disputed  statements  as  to  the  reac- 
tion of  the  muscles,  etc.  We  shall  only  refer  to  the  works  of  Rohmann,  PflTiger's  Arch., 
50  and  55,  and  Heffter,  Arch.  f.  exp.  Path.  u.  Pharm.,  31  and  38.  These  works  con- 
tain also  the  researches  of  the  older  investigators  more  or  less  completely. 

2  Untersuchungen  iiber  den  Stoffwechsel  der  Muskeln,  etc.,  Berlin,  1867. 

3  Amer.  Journ.  of  Physiol.,  9. 


METABOLISM    L\    THE    MUSCLES.  467 

as  Nasse  and  Werther,  have  obsen-ed  a  decrease  iii  the  quantity  of 
glycogen  in  rigor  of  the  muscle.  On  the  other  side,  Bohm  ^  has  observed 
cases  in  which  no  consumption  of  glycogen  took  place  in  rigor  of  the  muscle, 
and  he  has  also  found  that  the  quantity  of  lactic  acid  produced  is  not  pro- 
portional to  the  quantity  of  glycogen.  It  is  therefore  possible  that  the 
consumption  of  glycogen  and  the  formation  of  lactic  acid  in  the  muscles 
are  two  processes  independent  of  each  other,  and,  as  above  stated  in  regard 
to  the  formation  of  paralactic  acid,  the  lactic  acid  of  the  muscle  may  be 
considered  as  a  decomposition  product  of  protein.  The  origin  of  the  carbon 
dioxide  is  also  not  to  be  sought  for  in  the  decomposition  of  the  glycogen  or 
dextrose.  Pfluger  and  Stixtzixg  -  have  found  that  in  the  muscle  a  sub- 
stance occurs  which  evolves  large  quantities  of  car';on  dioxide  on  boiling 
with  water,  and  it  is  probably  this  substance  which  is  decomposed  with  the 
formation  of  carbon  dioxide  in  tetanus  as  well  as  in  rigor.  In  this  connec- 
tion we  call  attention  to  the  fact  that  phosphocarnic  acid  yields  lactic  acid 
as  well  as  carbon  dioxide  as  cleavage  products. 

After  the  muscles  have  been  rigid  for  some  time  tliey  relax  again  and 
become  softer.  This  is  in  part  produced  by  the  strong  acid  dissoh-ing  the 
myosin-clot  and  m  part  Ijy  autolx-tic  px"ocesses  (Vogel^). 

Metabolism  in  the  Inactive  and  Active  Muscles.  It  is  admitted  by  a 
nimiber  of  prominent  investigators,  Pfltger  and  Colasanti,  Zuntz  and 
RoHRiG.-*  and  others,  that  the  metabolism  in  the  muscles  is  regulated  by 
the  nen'ous  system.  "\Mien  at  rest,  when  there  is  no  mechanical  exertion, 
there  exists  a  condition  which  Zuxtz  and  Rohrig  have  designated  ''chemical 
tonus."  This  tonus  seems  to  be  a  reflex  tonus,  for  it  may  be  reduced 
by  discontinuing  the  connection  between  the  muscles  and  the  central 
organ  of  the  nen^ous  system  by  cutting  through  the  spinal  cord  or  the 
muscle-ner\-es.  The  possibility  of  reducing  the  chemical  tonus  ol  the 
muscles  in  various  ways  offers  an  important  means  of  decidmg  the  extent 
and  kind  of  chemical  processes  going  on  in  the  muscles  when  at  rest.  In 
comparative  chemical  investigation  of  the  processes  in  the  active  and  the 
inactive  muscles  several  methods  of  procedure  have  been  adopted.  The 
same  active  and  inactive  muscles  have  been  compared  after  removal,  also 
the  arterial  and  venous  muscle-blood  in  rest  and  activity,  and  lastly  the 
total  exchange  of  material,  the  receipts  and  expenditures  of  the  organism, 
have  been  investigated  under  these  two  conditions. 

'Nasse,  Beitr.  z.  Physiol,  der  kontrakt.  Substanz,  Pfiuger's  Arch.,  2;  Werther, 
ibid.,  46;    Bohm,  ibid.,  23  and  46. 

2  Pfl  iger's  Arch.,  IS. 

^  R.  Vogel,  Unters.  iiber  Muskelsaft,  Deutsch.  Arch,  f .  klin.  Med.,  1902. 

*  See  the  works  of  Pfluger  and  his  pupils  in  Pfl^iger's  Arch.,  4,  12,  14,  16,  and  IS; 
Rohrig,  ibid.,  4.  See  also  Zuntz,  ibid.,  12-.  In  regard  to  the  metabolism  after  curare 
poisoning,  see  also  Frank  and  Voit,  Zeitschr.  f.  Biologic,  42,  and  Frank  and  Geb- 
hard,  ibid.,  43. 


468  .     MUSCLES. 

By  investigations  according  to  these  several  methods  it  has  been  found 
that  the  active  muscle  takes  up  oxygen  from  the  blood  and  returns  to  it 
carbon  dioxide,  and  also  that  the  quantity  of  oxygen  taken  up  is  greater 
than  the  oxygen  contained  in  the  carbon  dioxide  eliminated  at  the  same 
time.  The  muscle,  therefore,  holds  in  some  form  of  combination  a  part  of 
the  oxygen  taken  up  while  at  rest.  During  activity  the  exchange  of  mate- 
rial in  the  muscle,  and  therewith  the  exchange  of  gas,  is  increased.  The 
animal  organism  takes  up  much  more  oxygen  in  activity  than  when  at 
rest,  and  eliminates  also  considerably  more  carbon  dioxide.  The  quan- 
tity of  oxygen  which  leaves  the  body  as  carbon  dioxide  during  activity 
is  much  larger  than  the  quantity  of  oxygen  taken  up  at  the  same  time; 
and  the  venous  muscle-blood  is  poorer  in  oxygen  and  richer  in  carbon 
dioxide  during  activity  than  during  rest.  The  exchange  of  gases  in  the 
muscles  during  activity  is  the  reverse  of  that  at  rest,  for  the  active  muscle 
gives  up  a  quantity  of  carbon  dioxide  which  does  not  correspond  to  the 
quantity  of,  oxygen  taken  up,  but  is  considerably  greater.  It  follows  from 
this  that  in  muscular  activity  not  only  does  oxidation  take  place,  but  also 
splitting  processes  occur.  This  results  also  from  the  fact  that  removed 
blood-free  muscles  when  placed  in  an  atmosphere  devoid  of  oxygen  can 
labor  for  some  time  and  also  yield  carbon  dioxide  (Hermann  i). 

During  muscular  inactivity,  in  the  ordinary  sense,  a  consumption  of 
glycogen  takes  place.  This  is  inferred  from  the  observations  of  several 
investigators  that  the  quantity  of  glycogen  is  increased  and  its  correspond- 
ing consumption  reduced  in  those  muscles  whose  chemical  tonus  is  reduced 
either  by  cutting  through  the  nerve  or  for  other  reasons  (Bernard,  Chan- 
DELON,  Vay,2  and  others).  In  activity  this  consumption  of  glycogen 
is  increased,  and  it  has  been  positively  proved  by  the  researches  of  several 
investigators  (Nasse,  Weiss,  Kulz,  Marcuse,  Manche,  Morat  and 
DuFOUR  ^)  that  the  quantity  of  glycogen  in  the  muscles  in  activity  decreases 
quickly  and  freely.  As  shown  by  the  researches  of  Chauveau  and  Kauf- 
]vl\nn,  Quinquaud,  Morat  and  Dufour,  Cavazzani,  and  especially  those 
of  Seegen,"*  the  sugar  is  removed  from  the  blood  and  consumed  during 
activity.     According   to   Seegen    a   very    abundant   formation    of   sugar 

'  1.  c.  In  regard  to  gas  exchange  in  removed  muscles,  see  also  J.  Tissot,  Arch,  de 
Physiol.  (5),  6  and  7,  and  Compt.  rend.,  120. 

^  Chandelon,  Pfliiger's  Arch.,  13;  Vay,  Arch.  f.  exp.  Path.  u.  Pharm.,  34,  which 
also  contains  the  pertinent  literature. 

^  Nasse,  Pfl  ger's  Arch.,  2;  Weiss,  Wien.  Sitzungsber.,  64;  Kiilz,  in  Ludwig's 
Festschrift,  Marburg,  1890;  Marcuse,  Pfl  iger's  Arch.,  39;  Manch^,  Zeitschr.  f.  Biolo- 
gic, 2.'>;   Morat  and  Dufour,  Arch,  de  Physiol.  (5),  4. 

^Chauveau  and  Kaufmann,  Compt.  rend.,  103,  104,  and  105;  Quinquaud,  Maly's 
Jahresber.,  10;  Morat  and  Dufour,  1.  c;  Cavazzani,  Centralbl.  f.  Physiol.,  8;  Seegen, 
"Die  Zuckerbildung  im  Thierkorper,"  Berlin,  1890,  Centralbl.  f.  Physiol.,  S,  9,  and 
10;  Arch.  f.  (Anat.  u.)  Physiol.,  1895  and  1896;   Pfliiger's  Arch.,  50. 


ACTIVITY  AND  FORMATION  OF  ACID.  469 

takes  place  in  the  liver,  and  correspondingly  the  blood  of  the  hepatic  vein 
is  much  richer  in  sugar  than  that  in  the  portal  vein;  and  this  sugar  of  the 
blood  is,  according  to  him,  the  source  of  heat  formation  and  mechanical 
activity.  It  is  nevertheless  true  that  important  objections  have  been 
presented  against  a  few  of  these  investigations,  and  a  sugar  formation, 
according  to  Seegex's  idea,  has  been  denied  by  several  investigators, 
and  recently  by  Ztjntz  and  Mosse;  but  still  there  can  exist  hardly  any 
doubt  that  sugar  is  consumed  in  muscular  activity.  A  direct  proof  for  this 
has  recently  been  given  by  Joh.  Muller.^  In  experiments  on  surva\Tiig 
cats'  hearts  which  were  percolated  with  a  salt  solution  containing  sugar, 
he  could  detect  an  undoubted  consumption  of  sugar  which  was  quite  con- 
siderable. 

The  amphoteric  reaction  of  the  inactive  muscles  is  changed  during 
activity  to  an  acid  reaction  (Du  Bois-Reymoxd  and  others),  and  the  acid 
reaction  increases  to  a  certain  point  with  the  work.  The  quickly  contract- 
ing pale  muscles  produce,  according  to  Gleiss,^  more  acid  during  acti-vity 
than  the  more  slowly  contracting  red  muscles.  The  acid  reaction  appearing 
during  activity  was  formerly  considered  to  be  due  to  the  formation  of  lactic 
acid,  a  view  which  has  been  contradicted  by  Astaschewsky,  Pfluger.  and 
Waerex,  who  found  less  lactic  acid  in  the  tetanized  muscle  than  when  at 
rest.  MoxAEi  also  found  a  decrease  in  the  quantity  of  lactic  acid  during 
activity,  and  according  to  Heffter  the  quantity  of  lactic  acid  in  the  muscle 
is  diminished  in  tetanus  produced  by  poison.  Contrar}'  to  these  investiga- 
tions, Marcuse  and  Werther  have  been  able  to  prove  the  formation  of 
lactic  acid  during  activity;  still  the  statements  are  ven,'  contradictor}-. 
Other  observations  indicate  a  formation  of  lactic  acid  during  acti\'ity. 
Thus  Spiro  found  an  increase  in  the  quantity  of  lactic  acid  in  the  blood 
during  work.  Colasanti  and  Moscatelli  found  small  quantities  of  lactic 
acid  in  human  urine  after  strenuous  marches,  and  Werther  observed  an 
abundance  of  lactic  acid  in  the  urine  of  frogs  after  tetanization.  According 
to  Hoppe-Seyler,  on  the  contrary',  in  agreement  with  his  view  in  regard 
to  the  formation  of  lactic  acid,  lactic  acid  is  not  produced  regularly  during 
work,  but  only  when  insufficient  oxygen  is  supplied.  Zillesex  ^  has  also 
found  that  on  artificially  cutting  off  the  oxygen  from  the  muscles  during 
life  more  lactic  acid  was  formed  than  under  normal  conditions. 

1  Mosse,  Pfluger's  Arch.,  63;  Zuntz,  Centralbl.  f.  Thysiol.,  10,  and  Arch.  f.  (Anat. 
u)  Fhysiol.,  1896,  538.  See  also  Schenck,  Pfluger's  Arch.,  61  and  65;  Miiller, 
Zeitschr.  f.  allgem.  Physiol.,  3. 

2  Pfluger's  Arch.,  41. 

^Astaschewsky,  Zeitschr.  f.  physiol.  Chem.,  4;  Warren,  Pfliiger's  Arch.,  24; 
Monari,  Maly's  Jahresber.,  19;  Heffter,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Marcuse, 
1.  c;  Werther,  Pfluger's  Arch.,  46;  Spiro,  Zeitschr.  f.  physiol.  Chem.,  1;  Colasanti 
and  Moscatelli,  Maly's  Jahresber.,  17,  212;  Hoppe-Seyler,  1.  c,  and  Zeitschr.  f.  physiol. 
Chem.,  19;   Zillesen,  ibid.,  15. 


470  MUSCLES. 

It  is  evident  that  the  experiments  with  the  muscles  in  situ — in  other 
words,  with  muscles  through  which  blood  is  passing — cannot  yield  any  con- 
clusion to  the  above  question,  as  the  lactic  acid  formed  during  work  may 
j)erhaps  be  removed  by  the  blood.  The  following  objections  can  be  made 
against  those  experiments  in  which  lactic  acid  has  been  found  after  mod- 
erate work  in  the  blood  or  the  urine,  as  also  especially  against  the  experi- 
ments with  removed  active  muscles,  namely,  that  in  these  cases  the  supply 
■of  ox3'gen  to  the  muscles  was  not  sufficient,  and  that  the  lactic  acid  formed 
thereby  is  not,  in  accordance  with  the  views  of  Hoppe-Seyler,  a  perfectly 
normal  process.  The  question  as  to  the  formation  of  lactic  acid  in  the 
active  muscle  under  perfect  physiological  conditions  is  still  an  open  one, 
although  several  observations  make  it  seem  to  be  very  probable. 

According  to  Siegfried  the  amount  of  phosphocarnic  acid  is  dimin- 
ished during  activity.  Macleod  claims  that  this  is  true  only  for  intense 
muscular  activity,  while  with  ordinary  work  the  organic  phosphorus 
not  present  as  nucleons  is  diminished  and  the  quantity  of  phosphates 
is  increased.  Tliis  stands  in  accord  with  Weyl  and  Zeitler's  ^  observa- 
tions that  the  active  muscle  contains  more  phosphoric  acid  than  the  inac- 
tive muscle.  As  in  the  dead  muscle,  so  in  the  active  muscle,  the  some- 
what stronger  acid  reaction  is  in  part  due  to  a  greater  quantity  of  mono- 
phosphate. 

The  amount  of  protei  .s  in  the  removed  muscles  is,  according  to  the 
older  investigators,  decreased  by  work.  The  correctness  of  this  statement 
is,  however,  disputed  by  other  investigators.  The  older  statements  in 
regard  to  the  nitrogenous  extractive  bodies  of  the  muscle  in  rest  and  in 
activity  are  likewise  uncertain.  According  to  the  recent  researches  of 
MoNARi^  the  total  quantity  of  creatine  and  creatinine  is  increased  by 
work,  and  indeed  the  amount  of  creatinine  is  especially  augmented  by  an 
excess  of  muscular  activity.  The  creatinine  is  formed  essentially  from 
the  creatine.  In  excessive  activity  Monari  also  found  xanthocreatinine 
in  the  muscle,  and  the  quantity  was  one-tenth  that  of  the  creatinine.  The 
purine  bases  are,  according  to  Burian,^  increased  during  work,  due  to  a 
greater  formation  (see  above,  page  454).  It  seems  to  have  been  posi- 
tively shown  that  the  active  muscle  contains  a  smaller  quantity  of  bodies 
soluble  in  water  and  a  larger  quantity  of  bodies  soluble  in  alcohol  than  the 
resting  muscle.  (Helmholtz  *). 

Attempts  have  been  made  to  solve  the  question  relative  to  the  behavior 


'Siegfried,  Zeitschr.  f.  physiol.  Chem.,  21;    Macleod,  ibid.,  28;    Weyl  and  Zeitler, 
ibid.,  6. 

^'Maly's  Jahresber.,  19,  296. 
^  Zeitschr.  f.  physiol.  Chem.,  43. 
^  Arch.  f.  (Anat.  u.)  Physiol.,  1845. 


NITROGENOUS  METABOLISM   IN  THE   MUSCLES.  471 

of  the  nitrogenized  constituents  of  the  muscle  at  rest  and  during  activity  by 
determining  the  total  quantity  of  nitrogen  eliminated  under  these  different 
conditions  of  the  body.  AMiile  formerly  it  was  held  with  Liebig  that  the 
elimination  of  nitrogen  by  the  urine  was  increased  by  muscular  work,  the 
researches  of  several  experimenters,  especially  those  of  Voit  on  dogs  and 
Pettenkofer  and  Voit  on  men,  have  led  to  quite  different  results.  They 
have  shown,  as  has  also  lately  been  confirmed  by  other  investigators, 
especially  I.  Munk  and  Hirschfeld,i  that  during  work  no  increase  or 
only  a  very  insignificant  increase  m  the  elimination  of  nitrogen  takes  place. 

We  should  not  omit  to  mention  the  fact  that  a  series  of  experiments 
has  been  made  showing  a  significant  increase  in  the  metabolism  of  proteins 
during  or  after  work.  There  are  for  example  the  observ^ations  of  Flint 
and  of  Pavy  on  a  pedestrian,  v.  Wolff,  v.  Funke,  Kreuzhage,  and 
Kellxer  on  a  horse,  and  Dunlop  and  his  collaborators  on  working  human 
beings,  and  of  Krum^l\cher,  Pfluger,  Zuxtz  and  his  pupils,^  and  others. 
The  researches  on  the  elimination  of  sulphur  during  rest  and  activity  also 
belong  to  this  categoiy.  The  elimination  of  nitrogen  and  sulphur  runs 
parallel  with  the  metabolism  of  proteir.s  in  resting  and  active  persons,  and 
the  quantity  of  sulphur  excreted  by  the  urine  is  therefore  also  a  measure 
of  the  protein  catabolism.  The  older  researches  of  Exgelmanx,  Flint, 
and  Pavy,  as  well  as  the  more  recent  ones  of  Beck  and  Benedict,^  and 
Dunlop  and  his  collaborators,  show  an  increased  elimination  of  sulphur 
during  or  after  work,  and  this  indicates  an  increased  protein  metabolism 
because  of  muscular  activity. 

That  an  increased  destruction  of  protein  is  not  necessarily  produced  by 
work  follows  from  the  observations  of  Caspari,  Bornstein,  Kaup,  Wait, 
A.  LoEW^',  Atwater  and  Benedict,^  that  a  retention  of  nitrogen  and  a 
deposition  of  protein  occur  during  work.  The  contradictory^  observations 
on  the  protein  destruction  during  and  caused  by  work  are  not  directly  in 
opposition  to  each  other,  because  the  extent  of  protein  metabolism  is  de- 
pendent upon  man}'  conditions,  such  as  the  quantity  and  composition  of 
the  food,  the  condition  of  the  adipose  tissue  of  the  body,  the  action  of  the 

'  Voit,  Untersuchungen  uber  den  Eiiifluss  des  KcchsaLzes,  des  Kaffees  und  der 
Muskelbewegungen  auf  den  Stoffwechsel  Ol^inchen,  1860),  and  Zeitschr.  f.  Biologie,  2; 
J.  Munk,  Arch.  f.  (Anat.  u.)  Physiol.,  1890  and  1896;  Hirschfeld,  Virohow's  Arch.,  121. 

2  Flint,  Journ.  of  Anat.  and  Physiol.,  11  and  12;  Pa\y,  The  Lancet,  1876  and  1877; 
V.  Wolff,  V.  Funke,  Kellner,  cited  from  Voit,  Hermann's  Handb.,  6,  197;  Dunlop, 
Nool-Paton,  Stockman,  and  Maccadam,  Journ.  of  Physiol.,  22;  Krummacher,  Zeitschr. 
f.  Biologie,  33;  Pfl  ger,  Pfi  ger's  Arch.,  50;  Zuntz,  Arch.  f.  (Anat.  u.)  Physiol.,  1894. 

^  Engelmann,  Arch.  f.  (Anat.  u.)  Physiol.,  1871 ;  Beck  and  Benedict,  Pfitiger's  Arch., 
54,  and  also  foot-note  2. 

*  Caspari,  Pfl  ger's  Arch.,  83;  Bornstein,  ibid.;  Kaup,  Zeitschr.  f.  Biologie,  43; 
Wait,  U  S  Depart.  Agricult.  BulletinSO  (1901);  Atwater  and  Benedict,  ibid.,  Bull. 
69  (1899);   Loewj,  Arch.  f.  (Anat.  u.)  Physiol.,  1901. 


472  MUSCLES 

work  upon  the  respiratory  mechanism,  etc.,  all  of  which  have  an  influence 
on  the  results  of  the  experiments. 

Recently  Steyrer  ^  has  found  that  the  muscle  juice  of  a  continuously  tetanized 
muscle  was  somewhat  poorer  in  musculin  and  correspondingly  richer  m  myogen 
than  the  juice  from  a  similar  non-tetanized  muscle.  We  cannot  draw  any  con- 
clusions from  this  experiment,  but  it  seems  to  show  that  the  proteins  are  not 
consumed  in  work. 

The  older  investigations  on  the  amount  of  fat  in  muscles  removed  after 
activity  and  after  rest  have  not  led  to  any  definite  results.  According  to 
the  recent  investigations  of  Zuntz  and  Bogdanow,^  the  fat  belonging  to 
the  muscle-fibres  and  which  is  difficultly  extracted  takes  part  in  work. 
Besides  these  there  are  several  researches  by  Voit,  Pettenkofer  and  Voit, 
J.  Frentzel,3  and  others  which  make  an  increased  destruction  of  fat 
during  work  probable. 

If  the  results  of  the  investigations  thus  far  made  of  the  chemical  proc- 
esses going  0.1  in  the  active  and  inactive  muscle  were  collected  together,  we 
would  find  the  following  characteristics  for  the  active  muscle.  The  active 
muscle  takes  up  more  oxygen  and  gives  off  more  carbon  dioxide  than  the 
inactive  muscle;  still  the  elimination  of  carbon  dioxide  is  increased  con- 
siderably more  than  the  absorption  of  oxygen.     The  respirator}^  quotient^ 

CO 

-^^,  is  found  to  be  regularly  raised  during  work ;  yet  this  rise,  which  will 

be  explained  in  detail  in  a  following  chapter  on  metabolism,  can  hardly  be 
conditioned  on  the  kind  of  processes  going  on  in  the  muscle  during  activity 
with  a  sufficient  supply  of  oxygen.  In  work  a  consumption  of  carbo- 
hydrates, glycogen,  and  sugar  takes  place.  The  acid  reaction  of  the  muscle 
becomes  greater  with  work.  In  regard  to  the  extent  of  a  re-formation  of 
lactic  acid  opinion  is  divided.  An  increased  consumption  of  fat  has  occa- 
sionally been  observed.  The  quantity  of  organic  phosphorus  decreases, 
and  an  increase  in  the  nitrogenous  extractives  of  the  creatinine  group 
seems  also  to  occur.  Protein  metabolism  has  been  found  increased  in 
certain  series  of  experiments  and  not  in  others;  but  an  increased  elimina- 
tion of  nitrogen  as  a  direct  consequence  of  muscular  exertion  has  thus 
far  not  been  positively  proved. 

In  close  connection  with  the  above-mentioned  facts  there  is  the  question 
as  to  the  material  basis  of  muscular  activity  so  far  as  it  has  its  origin  in 
chemical  processes.  In  the  past  the  generally  accepted  opinion  was  that  of 
LiEBiG,  that  the  source  of  muscular  action  consisted  of  a  catabolism  of  the 
protein  bodies;  to-day  another  generally  accepted  view  prevails.     Fick  and 


Hofmeister's  Beitrage,  4.  ^  Arch.  f.  (Anat.  u.)  Physiol.,  1897. 

^  Pfl  iger's  Arch.,  68. 


SOURCE  OF   MUSCULAR  ENERGY.  473 

WisLiCENUS  ^  climbed  the  Faulhorn  and  calculated  the  amount  of  mechan- 
ical force  expended  in  the  attempt.  With  this  they  compared  the  mechan- 
ical equivalent  transformed  in  the  same  time  from  the  proteins,  calculated 
from  the  nitrogen  eliminated  with  the  urine,  and  found  that  the  work 
really  performed  was  not  by  any  means  compensated  by  the  consumption  of 
protein.  It  was  therefore  proved  by  this  that  proteins  alone  cannot  be  the 
source  of  muscular  activity,  and  that  this  depends  in  great  measure  on  the 
metabolism  of  non-nitrogenous  substances.  Many  other  observations  have 
led  to  the  same  result,  especially  the  experiments  of  Voit,  of  Pettenkofer 
and  Voit,  and  of  other  mvestigators,  whose  observations  show  that  while 
the  elimination  of  nitrogen  remains  unchanged,  the  elimination  of  carbon 
dioxide  during  work  is  ver}-  considerably  increased.  It  is  also  generally 
considered  as  positively  proved  that  muscular  work  is  produced,  kt  least  in 
greatest  part,  by  the  cataboUsm  of  non-nitrogenous  substances.  Never- 
theless there  is  no  warrant  for  the  statement  that  muscular  activity  is  pro- 
duced entirely  at  the  cost  of  the  non-nitrogenous  substances,  and  that  the 
protein  bodies  are  without  importance  as  a  source  of  energy. 

The  investigations  of  Pflltger^  are  of  great  interest  in  this  connection. 
He  fed  a  bulldog  for  more  than  seven  months  with  meat  which  alone  did 
not  contain  sufficient  fat  and  carbohj^lrates  even  for  the  production  of 
heart  activity,  and  then  let  him  work  very  hard  for  periods  of  14,  35,  and  41 
days.  The  positive  result  obtained  by  these  series  of  experiments  was  that 
"complete  muscular  activity  may  be  effected  to  the  greatest  extent  in  the 
absence  of  fat  and  carbohydrates,"  and  the  ability  of  proteins  to  serve  as  a 
source  of  muscular  energy  cannot  be  denied. 

The  nitrogenous  as  well  as  the  non -nitrogenous  nutriments  may  serve  as 
a  source  of  energy;  but  the  views  are  divided  in  regard  to  the  relative 
value  of  these.  PflCger  claims  that  no  muscular  work  takes  place  without 
a  decomposition  of  protein,  and  the  living  cell-substance  prefers  always 
the  protein  and  rejects  the  fat  and  sugar,  contenting  itself  with  these  only 
when  proteins  are  absent.  Other  investigators,  on  the  contrar}%  believe 
that  the  muscles  first  draw  on  the  supply  of  non-nitrogenous  nutriments, 
and  according  to  Seegen,  Chauveau,  and  Laulanie  ^  the  sugar  is  ir.deed 
the  only  direct  source  of  muscular  force.  The  last-mentioned  investigator 
holds  that  the  fat  is  not  directly  utilized  for  work,  but  only  after  a  previous 
conversion  into  sugar.  Zuntz  and  his  collaborators  have  made  strong 
objections  to  the  correctness  of  such  a  view.  If,  according  to  Zuntz,  the 
fat  must  be  first  transformed  into  sugar  before  it  can  serve  as  the  source  of 

■  Vierteljahrsschr.  d.  Zurich,  naturf.  Gesellsch.,  10,  cited  from  Centralbl.  f.  d. 
med.  Wiss.,  1866,  309. 

'  Pfliger's  Arch.,  50. 

^  See  Seegen,  foot-note  4,  page  468.  The  works  of  Chauveau  and  his  collaborators 
are  found  in  Compt.  rend.,  121,  122,  and  123;  Laulanie?,  Arch,  de  Physiol.  (5),  8. 


474  MUSCLES. 

muscular  work,  a  definite  expenditure  of  force  must  require  about  30  per 
cent  more  energy  wdth  fatty  food  than  it  does  with  carbohydrates;  but  this 
is  not  the  case.  The  investigations  of  Zuntz,  (together  with)  Loeb,  Heine- 
MANN,  Frextzel  and  Reach  show  that  all  foodstuffs  have  nearly  the  same 
power  of  serving  as  the  material  for  the  work  of  the  muscles.  The  extensive 
metabolism  investigations  of  Atwater  and  Benedict  ^  have  also  led  to 
similar  results  as  to  the  fats  being  a  source  of  muscular  energy.  The  law 
of  the  substitution  of  the  foodstuffs,  according  to  their  combustion  equiva- 
lents, is  also  true  for  muscular  work,  and  fat  correspondingly  acts  with  its 
full  amount  of  energy  without*  previously  being  transformed  into  sugar. 
The  question  which  of  the  foodstuffs  the  muscle  prefers  is  dependent  upon 
the  relative  quantities  of  the  same  at  the  disposal  of  the  muscle.  A  direct 
substitution  of  the  body  material  by  the  bodies  supplied  as  food  does  not 
take  place  in  the  muscular  activity  in  the  ordinary  nutritive  condition. 
According  to  Johansson  and  Koraen  -  the  CO2  excretion  produced  by 
certain  work  is  not  influenced  by  the  supply  of  foodstuffs  (protei    or  sugar). 

Siegfried  considers,  as  above  stated,  the  phosphocarnic  acid  as  a  source  of 
energy.  According  to  his  and  KRiJGER's  ^  researches,  phosphocarnic  acid,  which 
yields  on  cleavage,  among  other  bodies,  carbon  dioxide,  occurs  in  part  preformed 
in  the  muscle,  and  in  part  as  a  hypothetical  aldehyde  compound  of  the  same — a 
compound  which  forms  phosphocarnic  acid  on  oxidation.  Siegfried  therefore 
makes  the  suggestion  that  in  the  resting  muscle,  which  requires  more  oxygen 
than  exists  in  the  carbon  dioxide  ehminated,  this  reducing  aldehyde  substance  is 
gradually  oxidized  to  phosphocarnic  acid,  which  is  used  in  the  acti^^ty  of  the 
muscle  with  the  splitting  off  of  carbon  dioxide. 

Quantitative  Composition  of  the  Muscle.  A  large  number  of  analyses 
have  been  made  of  the  flesh  of  various  animals  for  purely  practical  purposes, 
in  order  to  determine  the  nutritive  value  of  different  varieties  of  meat;  but 
there  are  no  exact  scientific  analyses  with  sufficient  regard  to  the  quantity  of 
different  protein  bodies  and  the  remaining  muscle  constituents,  that  is, 
these  analyses  are  incomplete  or  of  little  value. 

To  give  the  reader  some  idea  of  the  variable  composition  of  muscje- 
substance  the  following  summary  is  presented,  chiefly  obtained  from  K.  B. 
Hofmann's  ■'  book,  although  it  does  not  correspond  to  the  present  demands. 
The  figures  are  parts  per  1000. 


'  Loeb,  Arch.  f.  (Anat.  u.)  Physiol.,  1894;  Heinemann,  Pfliiger's  Arch.,  83;  Frentzel 
and  Reach,  ibid.;  Atwater  and  Benedict,  U.  S.  Dept.  of  Agric,  Bull.  136,  and  Ergeb- 
ni.sse  der  Physiologie,  3. 

^Skand.  Arch.  f.  Physiol.,  13. 

2  Zeitschr.  f.  physiol.  Chem.,  22. 

*  Lehrbuoh  d.  Zoochemie  (Wien,  1876),  104. 


QU.\XTITATI\^E    COMPOSITION   OF    MUSCLES. 


475 


Muscles  of 

Maniiuals. 

Solids 217-255 

Water 745-783 

Organic  bodies 208-245 

Inorganic  bodies 9-10 

Myosin 35-106 

Stroma  substance  (Da^'ii.ewsky) 78-161 

Creat  ine 2 

Xanthine  bodies 1 . 3-1 . 7 

Inosinic  acid  (barium  salt) 0.1 

Protic  acid ■ — 

Taurine 0.7  (horse) 

Inosite 0.03 

Glycogen 4-37 

Lactic  acid 0.4-0.7 

Phosphoric  acid 3 . 4—4 . 8 

Potash 3.0-4.0 

Soda 0.3 

Lime 0.2 

Magnesia 0.4 

Sodium  chloride 0 .04-0 . 1 

Iron  oxide 0  .04-0  . 1 


Muscles  of 
Birds. 

Muscles  of 

Cold-blooded 
Animals. 

225-282 

200 

717-773 

800 

217-263 

180-190 

10-19 

10-20 

29.8-111 

29.7-87 

88.0-184 

70.0-121 

3.4 

2.3 

0.7-1.3 

— 

0.1-0.3 

— 

— 

7.0 

— 

1.1 

3-5 


In  this  table,  which  has  little  value  because  of  the  variation  in  the 
composition  of  the  muscles,  no  results  are  given  as  to  the  estimates  of  fat. 
O-uing  to  the  variable  quantity  of  fat  in  meat  and  the  incompleteness  of  the 
older  methods  of  estimation  it  is  hardly  possible  to  quote  a  positive  aver- 
age for  this  substance.  After  most  careful  efforts  to  remove  the  fat  from 
the  muscles  without  chemical  means,  it  has  been  fomid  thr.t  a  variable 
quantity  of  intermuscular  fat.  which  does  not  really  belong  to  the  muscular 
tissue,  always  remauis.  The  smallest  quantity  of  fat  in  the  muscles  from 
lean  oxen  is  6.1  p.  m.  according  to  Grouvex.  and  7.6  p.  m.  according  to 
Petersen.  This  last  observer  also  found  regularly  a  smaller  quantity  of 
fat,  7.6-8.6  p.  m.,  in  the  fore  quarters  of  oxen,  and  a  greater  amount, 
30.1-34.6  p.  m.,  in  the  hind  quarters  of  the  animal,  but  this  could  not  be 
substantiated  by  Steil.i  A  small  quantity  of  fat  has  also  been  fomid  in 
the  muscles  of  uild  animals.  B.  Konig  ard  F.-vrwick  fomid  10.7  p.  m.  fat 
in  the  muscles  of  the  extremities  of  the  hare,  and  14,3  p.  m.  in  the  muscles 
of  the  partridge.  The  muscles  of  pigs  and  fattened  animals  are,  when  all 
the  adherent  fat  is  removed,  very  rich  in  fat,  amounting  to  40-90  p.  m. 
The  muscles  of  certain  fishes  also  contain  a  large  quantity  of  fat.  According 
to  Almen,  in  the  flesh  of  the  salmon,  the  mackerel,  and  the  eel  there  are 
contained  respectively  100,  164,  and  329  p.  m.  fat.^ 


»  See  Steil,  Pfliiger's  Arch,,  61. 

^  In  regard  to  the  literature  and  complete  statements  on  the  composition  of  flesh 
of  various  animals,  see  Konig,  Chemie  der  menschUchen  Nahrungs-  und  Genussmittel, 
5.  Aufl. 


476  MUSCLES. 

The  quantity  of  water  in  the  muscle  is  liable  to  considerable  variation. 
The  quantity  of  fat  has  a  special  influence  on  the  quantity  of  water,  and 
one  finds,  as  a  rule,  that  the  flesh  which  is  deficient  in  water  is  correspond- 
ingly rich  in  fat.  The  quantity  of  water  does  not  depend  alone  upon  the 
amount  of  fat,  but  upon  many  other  circumstances,  among  which  must 
be  mentioned  the  age  of  the  animal.  In  young  animals,  the  organs  in 
general,  and  therefore  also  the  muscles,  are  poorer  in  solids  and  richer  in 
water.  In  man  the  quantity  of  water  decreases  until  mature  age,  but 
increases  again  towards  old  age.  Work  and  rest  also  influence  the  quantity 
of  water,  for  the  active  muscle  contains  more  water  than  the  inactive. 
The  uninterruptedly  active  heart  should  therefore  be  the  muscle  richest 
in  water.  That  the  quantity  of  water  may  vary  independently  of  the 
amount  of  fat  is  strikingly  shown  by  comparing  the  muscles  of  different 
species  of  animals.  In  cold-blooded  animals  the  muscles  generally  have 
a  greater  quantity  of  water,  in  birds  a  lower.  The  comparison  of  the  flesh 
of  cattle  and  fish  shows  verj^  strikingly  the  different  amounts  of  water 
(independent  of  the  quantity  of  fat)  in  the  flesh  of  different  animals. 
According  to  the  analysis  of  Almen,i  the  muscles  of  lean  oxen  contain  15 
p.  m.  fat  and  767  p.  m.  water;  the  flesh  of  the  pike  contains  only  1,5 
p.  m,  fat  and  839  p.  m.  water. 

For  certain  purposes,  as,  for  example,  in  experiments  on  metabolism,  it 
is  important  to  know  the  elementar}^  composition  of  flesh.  In  regard  to 
the  quantity  of  nitrogen  we  generally  accept  Voit's  figure,  namely,  3,4 
per  cent,  as  an  average  for  fresh  lean  meat.  According  to  Nowak  and 
HuppERT^  this  quantity  may  var}^  about  0,6  per  cent,  and  in  more  exact 
investigations  it  is  therefore  necessary  to  specially  determine  the  nitrogen. 
Complete  elementary  analyses  of  flesh  have  been  made  with  great  care  by 
Argutinsky,  The  average  for  ox-flesh  dried  in  va  uo  and  free  from  fat 
and  with  the  glycogen  deducted  was  as  follows:  C  49.6;  H  6.9;  N  15.3; 
0  +  S  23.0;  and  ash  5.2  per  cent.  Kohler  found  as  an  average  for  water 
and  fat -free  beef  C  49.86;  H  6.78;  N  15.68;  0  +  S  22.3  per  cent,  which  are 
very  similar  results.  This  investigator  has  also  made  similar  analyses  of 
the  flesh  of  various  animals  and  has  determined  the  calorific  value  of 
the  ash-  and  fat -free  dried  meat  substance.  This  value  was,  per  gram  of 
substance,  5.599-5.677  Cal.  The  relationship  of  the  carbon  to  nitrogen, 
which  Argutinsky  calls  the  "flesh  quotient,"  is  on  an  average  3,54:1. 
From  Kohler's  analyses  the  average  for  beef  is  3.15:1  and  for  horse-flesh 
3.38:1.  According  to  Salkowski,  of  the  total  nitrogen  of  beef  77.4  per 
cent  was  insoluble  proteins,  10.08  per  cent  soluble  proteins,  and  12.52  per 

>  Nova  Act.  Reg.Soc.  Scient.  Upsal.,  Vol.  extr.  ord.,  1877;  alsoMaly's  Jahresber.,  7. 
^  Voit,  Zeitschr.  f.  Biologie,  1;    Huppert,  ibid.,  7;   Nowak,  Wien.  Sitzungsber.,  64, 
Abt.  2. 


NON-STRIATED   MUSCLES.  477 

cent  other  soluble  bodies.     Frextzel  and  Schreuer  ^  find  that  about  7.74 
per  cent  of  the  total  nitrogen  belongs  to  the  nitrogenous  extractives. 

There  exist  complete  investigations  by  Katz  ^  as  to  the  quantity  of  min- 
eral constituents  of  the  muscles  from  man  and  animals.  The  variation  in 
the  different  elements  is  considerable.  Pork  is  much  richer  in  sodium  as 
compared  ^^ith  potassium  than  other  kinds  of  meat.  The  quantity  of  mag- 
nesium is  greater,  and  often  considerably  greater,  than  calcium  in  all  kinds 
of  flesh  investigated,  with  the  exception  of  the  haddock,  the  eel,  and  the 
pike.  Beef  is  very  poor  in  calcium.  Potassium  and  phosphoric  acid  are 
the  most  abundant  mineral  constituents  of  all  flesh. 

Non-striated  Muscles. 

The  smooth  muscles  have  a  neutral  or  alkaline  reaction  (Du  Bois- 
Reymond)  when  at  rest.  During  acti\dty  they  are  acid,  which  is  inferred 
from  the  observations  of  Bernstein,  who  found  that  the  almost  continually 
contracting  sphincter  muscle  of  the  Anodonta  is  acid  during  life.  The 
smooth  muscles  may  also,  according  to  Heidenhaix  and  KfHNE,  pass  into 
rigor  mortis  and  thereby  become  acid.  A  spontaneous  but  slowly  coagulat- 
ing plasma  has  also  been  obsers-ed  in  several  cases. 

In  regard  to  the  proteins  of  the  smooth  muscles  we  have  the  older 
statements  of  Heidenhain  and  Hellwig;^  but  they  were  first  carefully 
studied  according  to  newer  methods  by  Munk  and  Velichi.'*  These  experi- 
menters have  prepared  a  neutral  plasma  from  the  gizzard  of  geese,  accord- 
ing to  V.  Fl'Rth's  method.  This  plasma  coagulated  spontaneously  at  the 
temperature  of  the  room,  although  slowly.  It  contained  a  globulin,  pre- 
cipitated by  dialysis,  which  coagulated  at  55-60°  C.  and  also  showed  cer- 
tain similarities  with  Kuhne's  myosin.  A  spontaneously  coagulating 
albumin,  which  differed  from  myogen  (v.  Furth)  by  coagulatini  at  45-50° 
C,  and  which  passes  by  spontaneous  coagulation  into  the  coagulated  mod- 
ification without  a  soluble  intermediate  product,  exists  in  still  greater 
quantities  in  this  plasma.  Alkali  albuminates  do  not  occur,  but  a  nuclco- 
•proteid  is  found,  which  exists  in  about  five  times  the  quantity  as  compared 
with  striated  muscles.  Nucleon  is,  according  to  Panella,^  a  normal  con- 
stituent of  smooth  muscles  and  occurs  in  larger  amounts  than  in  striated 
muscles. 

*  Argutinskj',  Pfliiger's  Arch..  55;  Kohler,  Zeitschr.  f.  physiol.  Chem.,  31;  Sal- 
kowski,  Centralbl.  f.  d.  med.  Wissensch.,  1894;  Frentzel  and  Schreuer,  Arch.  f.  (Anat 
u.)  Physiol.,  1902. 

2  Pfliiger's  Arch.,  63.     See  also  Schmey,  Zeitschr.  f.  physiol.  Chem.,  39. 
^  Du  Bois-Reymond  in  Nasse,  Hermann's  Handb.,  1 ,  339;  Bernstein,  iftid. ;  Heiden- 
hain, ibid.,  340,  with  Hellwig,  ibid.,  339;  Ki.hne,  Lehrbuch,  331. 

*  Munk  and  Velichi,  Centralbl.  f.  Physiol.,  12. 

*  Maly's  Jahresber.,  34. 


478  MUSCLES. 

Recent  investigations  of  Bottazzi  and  Cappelli,  Vincent  and  Lewis 
Vincent,  and  v.  Fi'-rth/  some  on  the  muscles  of  warm-blooded  and  some 
on  those  of  lower  animals,  have  led  to  somewhat  contradictory  results,  but 
they  substantiate,  as  a  whole,  the  observations  of  Munk  and  Velichi. 
Besides  the  nucleoproteids  the  smooth  muscles  contain  two  bodies  corre- 
sponding in  coagulation  temperature  to  musculin  and  myosinogen  (myogen, 
V.  Fl'rth),  but  they  are  not  identical  therewith.  Hamoglohin  occurs  in  the 
smooth  muscles  of  certain  animals,  but  is  absent  in  others.  In  the  smooth 
muscles  (in  certain  varietie  of  animals)  freatine,  creatinine,  taurin:,  inosite, 
glycogen,  and  lactic  acid  have  been  found.  The  mineral  constituents  show 
the  remarkable  fact  that  the  sodium  compounds  exceed  the  potassium 
com  o  ui  Is. 

Hexze  found  abundance  of  taurine  in  the  muscles  of  octopods,  5  p.  m.,  but 
no  creatine,  which,  according  to  Fremy  and  Valenciennes,^  occurs  in  the  muscles 
of  cephalopods.  He  also  found  no  glycogen  and  no  paralactic  acid,  but,  on  the 
contrary,  small  amounts  of  fermentation  lactic  acid.  The  muscles  of  octopods 
are  richer  in  mineral  bodies  than  the  muscles  of  vertebrates,  and  are  nearly  twice 
as  rich  in  sulphur  as  these. 

'  Bottazzi,  Centralbl.  f.  Physiol.,  15;  Vincent  and  Lewis,  Journ.  of  Physiol.,  26; 
Vincent,  Zeitschr.  f.  physiol.  Chem.,  34;  v.  F  rth,  ibid.,  31. 

^  Henze,  ibid.,  43;  Fremy  and  Valenciennes,  cited  from  Kuhnc's  Lehrbuch,  p.  333. 


CHAPTER  XII. 
BRAIX  AND  XERVES. 

Ox  account  of  the  difficulty  in  making  a  mechanical  separation  and 
isolation  of  the  different  tissue-elements  of  the  central  nervous  organ  and 
the  nerv^es,  we  must  resort  to  a  few  microchemical  reactions,  chiefly  to 
qualitative  and  quantitative  investigations  of  the  different  parts  of  the 
brain,  in  order  to  study  the  varied  chemical  composition  of  the  cells  and 
the  ner^'e-axes.  This  study  is  accompanied  with  the  greatest  difficulty; 
and  although  our  knowledge  of  the  chemical  composition  of  the  brain  and 
nerves  has  been  somewhat  extended  by  the  investigations  of  modem  times, 
still  it  must  be  admitted  that  this  subject  is  as  yet  one  of  the  most  obscure 
and  complicated  in  physiological  chemistry. 

Proteins  of  d  fJerent  kinds  have  been  shown  to  be  chemical  constituents 
of  the  brain  and  nerves,  and  these  are  representatives  of  the  same  chief 
groups  as  occur  in  the  protoplasm.  In  the  brain  there  occur  some  pro- 
teins which  are  insoluble  in  water  and  neutral  salt  solutions,  and  which 
resemble  the  stroma  substances  of  the  muscles  and  cells,  while  other  pro- 
teins are  soluble  in  water  and  neutral  salt  solutions.  Among  the  latter 
we  find  chiefly  nucleoproteids  and  globulins.  The  nucleoproteid  found  by 
Halliburton  and  also  by  Levexe^  in  the  gray  substance  contains  0.5 
per  cent  phosphorus  and  coagulates  at  55-60°.  Levexe  obtained  adenine 
and  guanine  but  no  hypoxanthhie  as  cleavage  products.  According  to 
Halliburtox  there  are  two  globulins,  namely,  the  neuroglobulin  a,  which 
coagulates  at  47°  or  at  50-53°  in  the  caf.e  of  birds,  and  the  neuroglobulin  /?, 
whose  coagulation  temperature  is  70-75°,  but  which  varies  somewhat  in  dif- 
ferent animals.  In  the  frog  still  another  protein  body  occurs,  which  coag- 
ulates at  a  still  lower  temperature,  about  40°.  It  must  be  remarked  that 
the  coagulation  temperature  of  a-globulin  corresponds  with  the  tempera- 
ture of  the  first  heat  contraction  of  tlie  nerves  of  different  classes  of  animals 
(Halliburtox). 

Just  as  there  are  lecithin-albumins,  compounds  of  proteid  \\ith  lecithin,  so 

'  Halliburton,  On  the  Chemical  Physiology  of  the  Animal  Cell,  King's  College, 
London,  Physiological  Laboralorj-,  Collected  Papers  No.  1,  1893,  and  Ergebnisse  der 
Physiologic,  4;   Levene,  Arch,  of  Neurology  and  Psychopalhology,  2  (1899). 

479 


480  BRAIN  AND   NERVES. 

according  to  Ulpiani  and  Lelli  ^  there  exists  an  analogous  compound  in  the 
brain  which  is  a  combination  between  a  protagon-Uke  substance  and  a  pseudo- 
nuclein. 

There  does  not  seem  to  be  any  doubt  that  the  proteins  belong  chiefly 
to  the  gray  substance  of  the  brain  and  to  the  axis-cylinders.  The  same 
remark  also  applies  to  the  nuclein,  which  v.  Jacksch  ^  found  in  large  quan- 
tities in  the  gray  substance.  Neurokeratin,  which  was  first  detected  by 
KtJHNE,  and  which  partly  forms  the  neuroglia,  and  as  a  double  sheath 
envelops  the  outside  of  the  nerve-medulla  under  Schwann's  sheath  and 
the  inner  axis-cylinders,  occurs  in  the  nerves,  but  chiefly,  or  according  to 
Koch  entirely,  in  the  white  substance  (Kuhne  and  Chittenden,  Baum- 

STARK  ^). 

The  phosphorized  substance  protagon  must  be  considered  as  one  of  the 
chief  constituents,  perhaps  the  only  constituent  (Baumstark),  of  the  white 
substance.  This  last-mentioned  substance,  if  we  keep  for  the  present  to 
the  most  carefully  studied  protagon — because  there  are  perhaps  several 
different  protagons — yields  as  decomposition  products  lecithin,  fatty  acids, 
and  a  nitrogenous  substance,  cerebrin.  It  is  difficult  to  state  whether  this 
last  body  also  exists  preformed  in  the  brain.  At  least  an  allied  substance, 
cerebron,  occurs  preformed  in  the  brain.  That  lecithin  also  is  pre-existent 
in  the  brain  and  nerves  can  hardly  be  doubted.  The  investigations  thus 
far  made  have  not  shown  decisively  whether  it  is  more  abundant  in  the 
gray  or  the  white  substance;  according  to  Koch  it  is  much  more  abundant 
in  the  white  substance.  Fatty  acids  and  neutral  fats  may  be  prepared  from 
the  brain  and  nerves;  but  as  these  may  be  readily  derived  from  a  decom- 
position of  lecithin  and  protagon,  which  exist  in  the  fatty  tissue  between 
the  nerve-axes,  it  is  difficult  to  decide  what  part  the  fatty  acids  and  neutral 
fats  play  as  constitu?nts  of  the  real  nerve-substance.  Cholesterin  seems 
chiefly,  and  according  to  Koch  perhaps  entirely,  to  occur  in  the  white 
substance.  Besides  these  substances  the  nerve-tissue,  especially  the  white 
substance,  contains  doubtless  a  number  of  other  constituents  not  well 
known,  and  among  which  are  several  containing  phosphorus.  Thudi- 
chum,*  who  has  made  thorough  investigations  of  the  brain  and  has  described 
a  great  number  of  brain  constituents,  has  given  the  name  phosphatides  to 
all  substances  of  the  brain  containing  the  phosphoric-acid  radical.  Those 
phosphatides  which  contain  only  one  phosphoric-acid  radical  are  called 
monophosphati4es,  those    with  two    such  radicals  diphosphatides.     The 

1  Cited  from  Chem.  Centralbl.,  1902,  2,  292. 

2Pfl:iger's  Arch.,  13. 

^  Koch,  Amer.  Journ.  of  Physiol.,  11;  Kiihne  and  Chittenden,  Zeitschr.  f.  Biologie 
26;   Baumstark,  Zeitschr.  f.  physiol.  Chem.,  9. 

*  Thudichum,  Die  chemische  Konstitution  des  Gehims  des  Menschen  und  der  Tiers, 
Tubingen,  1901. 


PROTAGON.  481 

monophosphatides  can  contain  one,  two,  or  more  nitrogen  atoms  in  their 
molecule,  while  there  are  also  nitrogen-free  monophosphatides.  Irrespective 
of  the  relation  between  phosphorus  and  nitrogen,  certain  phosphatides 
differ  from  the  lecithins  by  not  yielding  any  glycerophosphoric  acid.  These 
investigations  of  Thudichum  are  without  doubt  of  great  importance,  but 
as  they  have  not  been  repeated  we  cannot  enter  into  a  discussion  of  the 
bodies  described  by  him. 

By  allowing  water  to  act  on  the  contents  of  the  medulla,  round  or 
oblong  double-contoured  drops  or  fibres,  not  unlike  double-contoured 
nerves,  are  formed.  These  remarkable  formations,  which  can  also  be  seen 
in  the  medulla  of  the  dead  nerve,  have  been  called  "myeline  forms,'^  and 
they  were  formerly  considered  as  produced  from  a  special  body,  "myeline." 
Myeline  forms  may,  however,  be  obtained  from  other  bodies,  such  as 
impure  protagon,  lecithin,  fat,  and  impure  cholesterin,  and  they  depend 
upon  a  decomposition  of  the  constituents  of  the  medulla.  According 
to  Gad  and  Heymans  ^  myeline  is  lecithin  in  a  free  condition  or  in  loose 
chemical  combination. 

The  extractive  bodies  seem  to  be  almost  the  same  as  in  the  muscles. 
One  finds  creatine,  which  may,  however,  be  absent  (Baumstark),  xanthine 
bodies,  inosite,  choline,  paralactic  acid  (Moriya),  phosphocarnic  acid,  uric 
acid,  jecorin  (according  to  Baldi,^  in  the  human  brain),  and  the  diamine 
neuridine,  C5H14N2,  discovered  by  Brieger^  and  which  is  most  interesting 
because  of  its  appearance  in  the  putrefaction  of  animal  tissues  or  in  cultures 
of  the  typhoid  bacillus.  Under  pathological  conditions  leucine  and  urea 
have  been  found  in  the  brain.  Urea  is  also  a  physiological  constituent  of 
the  brain  of  cartilaginous  fishes. 

Of  the  above-mentioned  constituents  of  the  nerv'e-substance  protagon 
and  the  cerebrins  or  cerebrosides  must  be  specially  described. 

Protagon.  This  body,  which  was  discovered  by  Liebreich,  is  a  nitrog- 
enized  and  phosphorized  substance  whose  elementary  composition,  accord- 
ing to  Gamgee  and  Blankenhorn,  is  C  66.39,  H  10.69,  N  2.39,  and  P  1.068 
per  cent.  Baumstark  and  Ruppel  obtained  the  same  figures,  while  Lieb- 
reich found  an  average  of  2.80  per  cent  N  and  1.23  per  cent  P.  Kossel 
and  Freytag,  who  obtained  still  higher  figures  for  the  nitrogen,  namely, 
3.25  per  cent,  and  somewhat  lower  figures  for  the  phosphorus,  0.97  per 
cent,  foimd  some  sulphur,  an  average  of  0.51  per  cent,  regularly  in  the 
protagon.  Ruppel  also  found  some  sulphur,  but  in  such  small  quantity 
that  he  considered  it  as  a  contamination.     Cramer  has  prepared  by  an 


^Arch.  f.  (Anat.  u.)  Physiol.,  1890. 

■  Baldi,  Arch.  f.   (Anat.  u.)  Physiol.,  1887,  Suppl.;    Moriya,  Zeitschr.  f.  physiol. 
Cheni.,  43. 

^  Brieger,  Ueber  Ptomaine,  Berlin,  1885  and  1886. 


482  BRAIN   AND   NERVES. 

essentially  different  method  a  protagon  which  contained  sulphur  but  had 
in  other  respects  the  same  composition  as  that  analyzed  by  Gamgek 
and  Blankexhorx.  Posxer  and  Gies  obtained,  in  a  very  extensive 
investigation,  fractions  which  had  variable  compositions.  These  last 
investigators,  as  well  as  Thudichum,  Lesem,  Worxer  and  Thierfelder, 
and  KocH,i  are  therefore  of  the  opinion  that  protagon  does  not  exist  as  a 
chemical  indi\adual,  but  is  a  mixture,  essentially  of  cerebrins,  lecithin, 
and  cephalin.  The  somewhat  variable  elementary  composition  also  indi- 
cates the  fact  that  the  protagon  as  ordinaril}'  obtained  is  not  a  homogene- 
ous substance.  On  the  contrarj^,  the  assumption  that  protagon  is  only  a 
mixture  of  cerebrins  and  lecithin-like  bodies  is,  according  to  Hammarstex, 
very  improbable.  That  a  mixture  of  amorphous  or  very  difficultly  crys- 
taUizable  substances  produces  so  readily  such  a  beautifully  crystalline 
substance  as  protagon,  which  can  be  recrj'stallized  as  often  as  one  wishes, 
contradicts  the  ordmary  chemical  experience.  What  seems  more  probable 
is  that  the  so-called  protagon  is  a  crj^stalline  substance  which  can  be  purified 
from  other  substances,  perhaps  its  own  decomposition  products,  with  the 
ver}'  greatest  difficult3\ 

As  we  are  not  decided  whether  protagon  is  only  a  mixture  or  is  a  body 
contaminated  with  other  substances,  it  is  difficult  to  decide  as  to  how  far 
the  so-called  decomposition  products  exist  as  preformed  constituents  of 
the  mixture  or  whether  they  are  true  decomposition  products.  On  boiling 
with  bar\'ta-water  protagon  yields  the  decomposition  products  of  lecithin, 
namely,  fatty  acids,  glycerophosphoric  acid,  and  choline.  Kossel  and 
Freytag  found  indeed  three  cerebrosides,  namely,  cerebrix,  kerasin 
(liomocerebrin),  and  excephalix. 

On  boiling  with  dilute  mineral  acids  protagon  yields  among  other  sub- 
stances a  reducing  carbohydrate.  On  oxidation  with  nitric  acid  protagon 
yields  higher  fatty  acids. 

Protagon  appears,  wiien  drj^,  as  a  loose  white  powder.  It  dissolves  in 
alcohol  of  85  vols,  per  cent  at  45°  C,  but  separates  on  cooling  as  a  snow- 
white,  flaky  precipitate,  consisting  of  balls  or  groups  of  fine  crystalline 
needles.  It  decomposes  on  heating  even  below  100°  C.  It  is  hardly 
soluble  in  cold  alcohol  or  ether,  but  dissolves,  at  least  when  freshly  precipi- 
tated, in  ether  on  warming.  It  swells  in  little  water  and  partly  decomposes. 
With  more  w^ater  it  swells  to  a  gelatinous  or  pasty  mass,  which  with  much 
water  yields  an  opalescent  Hquid.  On  fusing  with  saltpetre  and  soda, 
alkali  phosphates  are  obtained. 

"  Gamgee  and  Blankenhorn,  Zcitschr.  f.  physiol.  Chem.,  3;  Baumstark,  1.  c; 
Ruppel,  Zeitschr.  f.  Biologic,  31;  Liebreich,  Annal.  d.  Chem.  u.  Pharm.,  134;  Kossel 
and  Freytag,  Zeitschr.  f.  physiol.  Chem.,  17;  Womer  and  Thierfelder,  ibid.,  30;  Lesem 
and  Gies,  Amer.  Journ.  of  Thysiol.,  8;  Thudichum,  1.  c;  Cramer,  Joum.  of  Physiol., 
31;   Posner  and  Gies,  Journ.  of  Biolog.  Chem.,  1. 


CEREBRIN.  483 

Protagon  is  prepared  in  the  following  way:  An  ox-brain  as  fresh  as 
possible,  with  the  blood  and  membranes  carefully  removed,  is  ground  fine 
and  then  extracted  for  several  hours  -^dth  alcohol  of  85  vols,  per  cent  at 
45°  C,  filtered  at  the  same  temperature,  and  the  residue  extracted  with 
warm  alcohol  until  the  filtrate  does  not  yield  a  precipitate  at  0°  C.  The 
several  alcoholic  extracts  are  cooled  to  0°  C.  and  the  precipitates  united  and 
completely  extracted  with  cold  ether,  wiiich  dissolves  the  cholesterin  and 
lecithin-like  bodies.  The  residue  is  now  strongly  pressed  between  filter- 
paper  and  allowed  to  dr}-  over  sulphuric  acid  or  phosphoric  anliydride.  It 
is  now  pulverized,  digested  with  alcohol  at  45°  C,  filtered,  and  slowiy 
cooled  to  0°  C.  The  crystals  which  separate  may  be  purified  when  necessary 
by  recr\-stallization. 

The  same  steps  are  taken  when  one  wishes  to  detect  the  presence  of  pro- 
tagon. 

On  decomposing  protagon  (or  the  protagons)  by  the  gentle  action  of 
alkalies  we  obtain  as  cleavage  products,  as  above  stated,  one  or  more  bodies 
which  Thudichu:m  has  embraced  under  the  name  cerehrosides.  The  cere- 
brosides  are  nitrogenous  substances  free  from  phosphorus,  which  yield  a 
reducing  variety  of  sugar  (galactose)  on  boiling  with  dilute  mineral  acids. 
On  fusing  with  potash  or  by  oxidation  with  nitric  acid  they  jield  higher 
fatty  acids — palmitic  or  stearic  acids.  The  cerebrosides  isolated  from  the 
brain  are  cerebrin,  kerasin,  encephalin,  and  cerebron,  but  it  must  be  re- 
marked that  there  is  no  doubt  but  that  sometimes  the  same  body  of  varying 
purity  has  received  different  names.  The  bodies  isolated  by  Kossel  and 
Freytag  from  pus,  and  called  pyosin  and  pyogenin,  also  belong  to  the 
cerebrosides. 

Cerebrin.  Under  this  name  W.  Muller  i  first  described  a  nitrogenous 
substance,  free  from  phosphorus,  wiiich  he  obtained  by  extracting  with 
boiling  alcohol  a  brain-mass  which  had  been  pre\iously  boiled  with  bar}i:a- 
water.  Following  a  method  essentially  the  same,  but  differing  somewhat, 
Geoghegax  2  prepared  from  the  brain  a  cerebrin  with  the  same  properties 
as  Muller's,  but  containing  less  nitrogen.  According  to  Parous  ^  the 
cerebrin  isolated  by  Geoghegan,  as  well  as  by  Muller,  consists  of  a  mix- 
ture of  three  bodies,  "cerebrin,"  "homocerebrin,"  and  "encephalin."  Kos- 
sel and  Freytag  isolated  two  cerebrosides  from  protagon  which  were 
identical  with  the  cerebrin  and  homocerebrin  of  Parous.  According  to 
these  investigators,  the  two  bodies  phrenosin  and  kerasin,  as  described 
b}"  Thudichu:m,  seem  to  be  identical  with  cerebrin  and  homocerebrin. 

Cerebrin,  according  to  Parous,  has  the  following  composition:  C  69.08, 
H  11.47,  N  2.13,  O  17.32  per  cent,  which  corresponds  with  the  analyses 
made  by  Kossel  and  Freytag.     No  formula  has  been  given  to  this  body. 

'  Annal.  d.  Chem.  u.  Pharm.,  105. 

^  Zeitschr.  f.  physiol.  Chem.,  3. 

^  Parcus,  Ueber  einige  neue  GehimstoSe,  Inaug.-Diss.  Leipzig,  1881. 


484  BRAIN   AND  NERVES. 

In  the  dry  state  it  forms  a  pure  white,  odorless,  and  tasteless  powder.  On 
heating  it  melts,  decomposes  gradually,  smells  like  burnt  fat,  and  bums 
with  a  luminous  flame.  It  is  insoluble  in  water,  dilute  alkalies,  or  baryta- 
water;  also  in  cold  alcohol  and  in  cold  or  hot  ether.  On  the  contrary,  it 
is  soluble  in  boiling  alcohol  and  separates  as  a  flaky  precipitate  on  cooling, 
and  this  is  found  to  consist  of  a  mass  of  balls  or  grains  on  microscopical 
examination.  Cerebrin  forms  a  compomid  with  baryta,  which  is  insoluble 
in  water  and  is  decomposed  by  the  action  of  carbon  dioxide.  Cerebrin 
dissolves  in  concentrated  sulphuric  acid,  and  on  warming  the  solution  it 
becomes  blood-red.  The  variety  of  sugar  split  off  on  boiling  with  mineral 
acids — the  so-called  brain-sugar — is,  according  to  Thierfelder,i  galactose. 
Kerasln  (according  to  Thudichum),  or  homocerebrin  (according  to 
Parous),  has  the  following  composition:  C  70.06,  H  11.60,  N  2.23,  and 
O  16.11  per  cent.  Encephalin  has  the  composition  C  68.40,  H  11.60, 
N  3.09,  and  0  16.91  per  cent.  Both  bodies  remain  in  the  mother-liquor 
after  the  impure  cerebrin  has  precipitated  from  the  warm  alcohol.  These 
bodies  have  the  tendency  of  separating  as  gelatinous  masses.  Kerasin  is 
similar  to  cerebrin,  but  dissolves  more  easily  in  warm  alcohol  and  also  in 
warm  ether.  It  may  be  obtained  as  extremely  fine  needles.  Encephalin 
is,  according  to  Parous,  a  transformation  product  of  cerebrin.  In  the 
perfectly  pure  state  it  crystallizes  in  small  lamellae.  It  swells  into  a  pasty 
mass  in  warm  water.  Like  cerebrin  and  kerasin,  it  yields  a  reducing  sub- 
stance (probably  galactose)  on  boiling  with  dilute  acid. 

The  cerebrins  are  generally  prepared  according  to  MtJLLER's  method. 
The  brain  is  first  stirred  with  baryta-water  until  it  appears  like  thin  milk, 
and  then  it  is  boiled.  The  insoluble  parts  are  removed,  pressed,  and 
repeatedly  boiled  with  alcohol,  which  is  filtered  while  boiling  hot.  The 
impure  cerebrin  which  separates  on  cooling  is  freed  from  cholesterin  and 
fat  by  means  of  ether  and  then  purified  by  repeated  solution  in  warm 
alcohol.  According  to  Parous  this  repeated  solution  in  alcohol  is  con- 
tinued until  no  gelatinous  separation  of  homocerebrin  or  encephalin  takes 
place. 

According  to  Geoghegan's  method  the  brain  is  first  extracted  with  cold 
alcohol  and  ether  and  then  boiled  with  alcohol.  The  precipitate  which 
separates  on  the  cooling  of  the  alcoholic  filtrate  is  treated  with  ether  and 
then  boiled  with  baryta-water.  The  insoluble  residue  is  purified  by  re- 
peated solution  in  boiling  alcohol. 

The  cerebrin  may  also  be  obtained  from  other  organs  by  employing  the 
above  methods.  The  quantitative  estimation,  when  such  is  desired,  may 
be  performed  in  the  same  way. 

KossEL  and  Freytag  prepare  cerebrin  from  protagon  by  saponifying  it 
in  methyl  alcohol  solution  with  a  hot  solution  of  caustic  baryta  in 
methyl  alcohol.  The  precipitate  is  filtered  off  and  decomposed  in  water  by 
carbon  dioxide  and  the  cerebrin  or  cerebroside  extracted  from  the  insoluble 
residue  with  hot  alcohol. 

'  Zeitschr.  f.  physiol.  Chem.,  14. 


CEREBRON   AND  CEPHALIN.  485 

Whether  the  above-described  cerebrins  are  chemical  individuals  or 
mixtures,  i.e.,  impure  sub  tances,  is  still  undecided.  The  purest  cerebrin 
or  cerebroside  thus  far  investigated  is  undoubtedly  Thierfelder's  cerebron, 
and  there  is  hardly  any  doubt  also  that  Muller's  cerebrin  consisted  essen- 
tially of  cerebron. 

Cerebron.  This  cerebiin,  isolated  by  Thierfelder  and  Worner  and 
then  especially  studied  by  Thierfelder,  was  first  isolated  by  Gamgee  and 
called  'pseudocerehrin  by  him.  Thudichum's  ^  phrenosin  seems  to  be  im- 
pure cerebron.  Cerebron  can  be  prepared  directly  from  the  brain  without 
saponification  with  barj-ta,  by  treatment  with  alcohol  containing  benzene 
or  chloroform  at  a  temperature  of  50°,  and  hence  it  is  considered  as  existing 
preformed  in  the  brain.  According  to  Thierfelder  cerebron  has  the 
formula  C48H93NO9;  it  melts  at  212°,  dissolves  in  warm  alcohol,  and 
separates  out  on  cooling.  From  proper  solvents  (acetone  containing 
chloroform)  it  may  be  separated  as  small  needles  or  plates.  If  cerebron  is 
suspended  in  85  per  cent  alcohol  at  a  temperature  of  50°  C.  it  balls  together 
in  amorphous  masses,  and  from  these  need  e-  and  leaf -shaped  crj-stals 
gradually  form.  Cerebron  also  yields  galactose,  and  it  can  be  split  by 
acids,  best  in  methyl  alcohol  containing  sulphuric  acid,  into  galactose, 
a  base  called  sphingosin  by  Thudichum,  and  cerebronic  acid  (Thudichum's 
neurostearic  acid).  The  cleavage  takes  place,  according  to  Thierfelder, 
as  follows:  C48H93N09  +  2H20  =  C25H5o03  (cerebronic  acid)+Ci7H35N02 
(sphingosin) +  C6H12O6  (galactose).  The  cerebronic  acid  consists  of  snow- 
white  crs'stals  which  are  soluble  in  alcohol  and  in  ether.  They  melt  at 
99-100°  and  give  a  crystalline  methyl  ester  which  melts  at  65°.  Sphin- 
gosin is  a  base  which  does  not  form  marked  crj'stals  and  which  is  insoluble 
in  water  and  ether,  but  gives  a  sulphate  which  is  soluble  in  chloroform  and 
in  warm  alcohol.  According  to  Thierfelder  and  Kitagaw^a,  sphingosin 
is  not  a  unit  substance.^ 

Cephalin  is  a  phosphatide  whose  formula,  based  upon  the  investigations 
of  Thudichum  and  Koch^  is  probably  C42H82NPO13.  The  views  of  these 
two  investigators  as  to  the  constitution  of  this  body,  which  is  difficult  to 
purify,  differ  ver}'  considerably.  According  to  Thudichum,  on  cleavage  it 
yields  neurine,  glycerophosphoric  acid,  stearic  acid,  and  a  specific  fatt}'  acid, 
cephalic  acid.  According  to  Koch  it  contains  on  the  contrar}^,  only  orie 
methyl  group  attached  to  nitrogen,  and  is  therefore  probably  dioxj^stearvd- 
monomethyl  lecithin.  Cephalin  is  amorphous  and  swells  up  in  water  like 
lecithin.  It  is  soluble  in  cold  ether,  glacial  acetic  acid,  and  chloroform,  but 
is  insoluble  in  acetone  and  in  alcohol,  either  cold  or  warm.     It  is  obtained 

'  Thierfelder  and  Worner,  Zeitschr.  f.  physiol.  Chem.,  30;   Thierfelder,  ibid.,  43,  44, 
and  4G;  Gamgee,  Text-book  of  Physiol.  Chem.,  London,  ISSO;  Thudichum,  1.  c. 
^Zeitschr.  f.  physiol.  Chem.,  4S. 
'  Thudichum,  1.  c;   Koch,  Zeitschr.  f.  physiol.  Chem.,  30. 


486  BRAIN  AND  NERVES. 

from  the  brain  after  dehydration  with  acetone  by  extractin'j;  with  ether  and 
precipitating  the  concentrated  ethereal  extract  with  alcohol.  The  cephalin 
is  perhaps  identical  with  the  myeline  substance  isolated  by  Zuelzer  ^  from 
the  brain. 

Bethe  ^  has  prepared  the  following  decomposition  products  from  the  brain 
of  the  horse  after  treatment  with  CuCL'  and  alkali :  aminocerebrinic-acid  glucoside, 
Ci^HgiOsN,  which  on  boiling  with  hydrochloric  acid  yields  cerebrinic  acid,  amino- 
cerebrinic-acid chloride,  and  a  hexose  (galactose  ?) ;  phrenin,  perhaps  identical 
with  Thudichum's  krinosin;  cerebrinic-phosphoric  acid,  and  a  stearic  acid  differ- 
ing somewhat  from  the  ordinary  one. 

Neuridine,  CHi^No,  is  a  non-poisonous  diamine  discovered  by  Brieger,  and 
which  was  obtained  by  him  in  the  putrefaction  of  meat  and  gelatine,  and  from 
cultures  of  the  typhoid  bacillus.  It  also  occurs  under  physiological  conditions 
in  the  brain,  and  as  traces  in  the  yolk  of  the  egg. 

Neuridine  dissolves  in  water  and  yields  on  boiling  with  alkalies  a  mixture  of 
dimethylamine  and  trimethylamine.  It  dissolves  with  difficulty  in  amyl  alcohol. 
It  is  insoluble  in  ether  or  absolute  alcohol.  In  the  free  state,  neuridine  has  a 
peculiar  odor,  suggesting  semen.  With  hydrochloric  acid  it  gives  a  compound 
crystallizing  in  long  needles.  With  platinic  chloride  or  gold  chloride  it  gives 
crystallizable  double  compounds  which  are  valuable  in  its  preparation  and  detec- 
tion. 

The  so-called  corpuscula  amylacea,  which  occur  on  the  upper  surface  of  the 
brain  and  in  the  pituitary  gland,  are  colored  more  or  less  pure  violet  by  iodine 
and  more  blue  by  sulphuric  acid  and  iodine.  They  consist,  perhaps,  of  the  same 
substance  as  certain  prostatic  calculi,  but  they  have  not  been  closely  investigated. 

Quantitative  Composition  of  the  Brain.  The  quantity  of  water  is  greater 
in  the  gray  than  in  the  white  substance,  and  greater  in  new-born  or  young 
individuals  than  in  adults!  The  brain  of  the  foetus  contains  879-926  p.  m. 
water.  According  to  the  observations  of  Weisbach  3  the  quantity  of 
water  in  the  several  parts  of  the  brain  (and  in  the  medulla)  varies  at  differ- 
ent ages.  The  following  figures  are  in  1000  parts— A  for  men  and  B  for 
women : 

20-30  Years.             30-50  Years.  50-70  Years.  70-94  Years. 

'"X      "~^  '"X  fiT"  A.  B.  A.  B. 

White  brain-substance..  695.6     682.9  683.1  703.1  701.9  689.6  726.1  722.0 

Gray             "                      833.6     826.2  836.1  830.6  838. U  838.4  847.8  839.5 

Gvri             784.7     792.0  795.9  772.9  796.1  796.9  802.3  801.7 

Cerebellum   788.3     794.9  778.7  789.0  787.9  784.5  803.4  797.9 

Pons  Varolii 734.6     740.3  725.5  722.0  720.1  714.0  727.4  724.4 

Medulla  oblongata 744.3     740.7  732.5  729.8  722.4  730.6  736.2  733,7 

Quantitative  analyses  have  also  been  made  of  the  ox-brain  by 
Petrowsky,^  and  of  the  brain  of  a  horse  by  Baumstark.  In  the  analysis 
of  Petrowsky  the  protagon  has  not  been  considered,  and  all  organic  phos- 
phorized  substances  were  calculated  as  lecithin.     On  these  groimds  these 


>  W.  Koch,  Zeitschr.  f.  physiol.  Chem.,  36;  Zuelzer,  ibid.,  27. 

2  Arch.  f.  exp.  Path.  u.  Pharm.,  48. 

3  Cited  from  K.  B.  Hofmann's  Lehrb.  d.  Zoochemie  (Wien,  1876),  121. 
*  Pfluger's  Arch.,  7. 


COMPOSITION  OF  THE  BRAIN.  487 

analyses  are  not  of  much  value  from  a  certain  standpoint.  In  Baumstaek'8 
analyses  the  gray  and  the  white  substance  could  not  be  sufficiently  sepa- 
rated, and  these  analyses,  on  this  account,  show  partly  an  excess  of  white 
and  partly  an  excess  of  gray  substance;  nearly  one-half  of  the  organic 
bodies,  chiefly  consisting  of  bodies  soluble  in  ether,  could  not  be  exactly 
analyzed.  Neither  of  these  analyses  gives  sufficient  explanation  of  the 
quantitative  composition  of  the  brain. 

The  analyses  made  up  to  the  present  time  give,  as  above  stated,  an 
unequal  division  of  the  organic  constituents  in  ihe  gray  and  white  sub- 
stance. In  the  analyses  of  Petrowsky  the  quantity  of  proteins  and  gela- 
tine-forming substances  in  the  gray  matter  was  somewhat  more  than  one- 
half,  and  in  the  white  about  one-quarter  of  the  solid  organic  substances. 
The  quantity  of  cholesterin  in  the  white  was  about  one-half,  and  in  the 
gray  substance  about  one-fifth  of  the  solid  bodies.  A  greater  quantity  of 
soluble  salts  and  extractive  bodies  was  found  in  the  gray  subtance  than 
in  the  white  (Baumstark).  The  following  analyses  of  Baumstark  give 
the  most  important  known  constituents  of  the  brain  calculated  in  1000 
parts  of  the  fresh,  moist  substance.  A  represents  chiefly  the  white,  and 
B  chiefly  the  gray  substance. 

A.  B. 

Water 695.35  769.97 

Solids 304.65  230.03 

Protagon 25.11  10.80 

Insoluble  protein  and  connective  tissue 50 .02  60  .79 

Cholesterin,  free 18.19  6.30 

"            combined 26.96  17.51 

Nuclein 2.94  1.99 

Neurokeratin 18 .93  10  .43 

Mineral  bodies 5.23  5.62 

The  remainder  of  the  solids  probably  consists  chiefly  of  lecithin  and 
other  phosphorized  bodies.  Of  the  total  amount  of  phosphorus  15-20 
p.  m.  belongs  to  the  nuclein,  50-60  p.  m.  to  the  protagon,  150-160  p.  m. 
to  the  ash,  and  770  p.  m.  to  the  lecithin  and  the  other  phosphorized  organic 
substances. 

As  shown  by  the  above  analysis  Bauivistark  differentiated  between  free 
and  combined  cholesterin.  He  believed  that  a  part  of  the  cholesterin  in 
the  brain  occurred  in  the  combined  state,  perhaps  as  an  ester;  this  view 
has  been  found  to  be  incorrect  by  the  recent  investigations  of  BtJNZ.  He 
obtained  from  the  brain  neither  esters  of  cholesterin  with  higher  fatty 
acids  nor  other  compounds  of  cholesterin  which  split  on  saponification. 
Tebb  1  has  also  found  only  free  cholesterin. 

The  analysis  of  the  brain  of  an  epileptic  made  by  Koch  ^  is  of  very 
great  interest.     We  cannot  enter  into  a  discussion  of  his  method  of  analysis. 

» B:;nz,  Zeitschr.  f.  physiol.  Chem.,  46;  Tebb,  Joum.  of  Physiol.,  34. 

^  Amer.  Journ.  of  Physiol.,  11;  Koch  and  Woods,  Joum.  of  Diol.  Chem.,  1. 


488  BRAIN  AND  NERVES. 

In  order  to  make  the  figures  found  comprehensible,  it  is  perhaps  necessary 
to  call  attention  to  a  few  points.  The  two  cerebrins,  phrenosin  and  kerasin, 
were  calculated  from  the  quantity  of  galactose  split  off.  The  quantity  of 
phosphatides,  designated  by  Koch  as  lecithans,  was  determined  from  the 
quantity  of  methyl  groups  split  off  by  hydriodic  acid  below  240°  plus  the 
quantity  of  true  lecithin  calculated  from  the  quantity  of  methyl  groups 
split  off  at  about  300°.  The  difference  between  the  quantity  of  lecithin 
and  the  total  quantity  of  lecithans  gave  the  amount  of  cephalin  and  myelin. 
The  nature  of  the  sulphurized  substance  is  unknown.  As  the  protagon, 
according  to  Koch,  is  a  mixture  of  various  substances,  no  results  as  to 
the  quantity  is  given.     The  other  figures  require  no  explanation. 

Corpus  Cort€X 

Callasuin  (prefrontal). 

Water 67.97  84.13 

Protein 3.20  5.00 

Nucleoproteids 3.70  3.00 

Neurokeratin 2.70  (Chittenden)      0 .40  (Chittenden) 

Extractives  (water-soluble).  1.51  1.58 

Lecithins 5.19  3.14 

Cephalin  and  myelin 3.49  0.74 

Phrenosin  and  kerasin 4 .  57  1 .  55 

Cholesterin 4.86  0.70 

Sulphurized  substance 1 .40  1 .45 

Mineral  bodies 0.82  0.87 

As  the  cerebrosides  occur  chiefiy  in  the  myelin  sheath,  Koch,  starting 
from  the  amount  in  the  investigated  part  of  the  brain,  attempts  to  calculate 
the  amount  of  the  anah'zed  cortical  substance  in  the  white  nerves,  and 
on  the  basis  of  these  calculations,  he  finds  the  following  values  for  the  pure 
gray  substance,  free  from  nerve-fibres,  and  compares  them  with  the  corpus 
callosum.     The  results  are  in  100  parts  of  the  dry  substance. 

Corpus         Gray  Substance 

Callosum.  Al'^^  f[°™      , 

white  substance). 

Protein 10.00  21.70 

Nucleoproteids 11.56  9.66 

Neurokeratin 8 .  40  — 

Extractives 4.75  5.92 

Lecithins 16.22  7.67 

Cephalin  and  myelin 10 .  91  — 

Phrenosin  and  kerasin 14 .  29  — 

Cholesterin 15 .  20  — 

Sulphurized  substance 4 .  37  5 .  43 

According  to  Noll  the  white  substance  of  the  spinal  marrow  is  some- 
what richer  in  protagon  than  the  brain,  and  in  nerve  degeneration  the 
quantity  of  protagon  diminishes.  The  method  used  by  him  would  not 
allow  of  an  exact  determination  of  the  protagon.  Mott  and  Halliburton  ^ 
have  also  shown  that  in  degenerative  diseases  of  the  nervous  system  the 
quantity  of  substances  containing  phosphorus  diminishes  and  that  in  these 

'Noll,  Zeitschr.  f.  phy.siol.  Chem.,  27;  Mott  and  Halliburton,  Philos.  Transact., 
Ser.  B,  191  (1899)  and  194  (1901). 


TISSUES  AND  FLUIDS  OF  THE   EYE.  489 

cases,  especially  in  general  paralysis,  choline  passes  into  the  cerebrospinal 
fluid  and  the  blood.  In  degenerated  nerves,  the  quantity  of  water  increases 
and  the  phosphorus  decreases. 

The  quantity  of  neurokeratin  in  the  nerves  and  in  the  different  parts  of 
the  brain  has  been  carefiUly  determined  by  Kuhxe  and  Chittendex.i 
They  found  3.16  p.  m.  in  the  plexus  brachialis,  3.12  p.  m.  in  the  cortex  of 
the  cerebellum,  22.434  p.  m.  in  the  white  substance  of  the  cerebrum,  25.72- 
29.02  p.  m.  in  the  white  substance  of  the  corpus  callosum,  and  3.27  p.  m. 
m  the  gray  substance  of  the  cortex  of  the  cerebrum  (when  free  as  possible 
from  white  substance).  The  white  is  decidedly  richer  in  neurokeratin  than 
the  peripheral  nerves  or  the  gray  substance.  According  to  Griffiths,^ 
neurochitin  replaces  neurokeratin  in  insects  and  Crustacea,  the  quantity  of 
the  first  being  10.6-12  p.  m. 

The  quantity  of  mineral  constituents  in  the  brain  amounts  to  2.95-7.08 
p.  m.  according  to  Geoghegax.  He  fomid  in  1000  parts  of  the  fresh, 
moist  brain  0.43-1.32  CI;  0.956-2.016  PO4;  0.244-0.796  CO3;  0.102- 
0.220  SO4;  0.01-0.098  Feo(P04)2;  0.005-0.022  Ca;  0.016-0.072  Mg;  0.58- 
1.778  K;  0.450-1.114  Na.  The  gray  substance  yields  an  alkaline  ash,  the 
white  an  acid  ash. 

Appendix. 

THE  TISSUES  AND    FLUIDS   OF  THE   EYE. 

The  retina  contains  in  all  865-899.9  p.  m.  water,  57.1-84.5  p.  m.  protein 
bodies — myosin,  albumin,  and  mucin  (?),  9.5-28.9  p.  m.  lecithin,  and 
8.2-11.2  p.  m.  salts  (Hoppe-Seyler  and  Cahx  3).  The  mineral  bodies 
consist  of  422  p.  m.  Na2HP04  and  352  p.  m.  NaCl. 

Those  bodies  which  form  the  different  segments  of  the  rods  and  cones 
have  not  been  closely  studied,  and  the  greatest  interest  is  therefore  con- 
nected with  the  coloring-matters  of  the  retina. 

Visual  purple,  also  called  rhodopsin,  erythropsin,  or  visual  red,  is  the 
pigment  of  the  rods.  Boll  ^  observed  in  1876  that  the  layer  of  rods  in  the 
retina  during  life  had  a  purplish-red  color  which  was  bleached  by  the  action 
of  light.  KtJHNE  ^  showed  later  that  this  red  color  might  remain  for  a  long 
time  after  the  death  of  the  animal  if  the  eye  was  protected  from  daylight 
or  investigated  by  a  sodium  light.  Under  these  conditions  it  was  also 
possible  to  isolate  and  closely  study  this  substance. 

'  Zeitschr.  f.  Biologie,  26. 

'  Compt.  rend..  115. 

^Zeitschr.  f.  physiol.  Chem.,  5. 

*  Monatsschr.  d.  Kgl.  Preuss.  Akad.,  12.  Nov.,  1876. 

^  The  investigations  of  Klihne  and  his  pupils,  Ewald  and  Ayres,  on  the  visual  purple 
will  be  found  in  Untersuchungen  aus  dem  physiol.  Institut  der  Universitat  Heidel- 
berg, 1  and  2;  and  in  Zeitschr.  f .  Biologie,  32. 


490  BRAIN  AND  NERVES. 

Visual  red  (Boll)  or  visual  purple  (Kuhxe)  has  become  known  mainly 
by  the  investigations  of  Kijhne.  The  pigment  occurs  chiefly  in  the  rods 
and  only  in  their  outer  parts.  In  animals  whose  retina  has  no  rods  the 
visual  purple  is  absent,  and  is  also  necessarily  absent  in  the  macula  lutea. 
In  a  variety  of  bat  (Rhinolophus  hipposideros),  in  hens,  pigeons  and  new- 
born rabbits,  no  visual  purple  has  been  found  in  the  rods. 

A  solution  of  visual  purple  in  water  which  contains  2-5  per  cent  crys- 
tallized bile,  which  is  the  best  solvent  for  it,  is  purple-red  in  color,  quite 
clear,  and  not  fluorescent.  On  evaporating  this  solution  in  vacuo  we 
obtain  a  residue  similar  to  ammonium  carminate  which  contains  violet  or 
black  grains.  If  the  above  solution  is  dialyzed  with  water,  the  bile  diffuses 
and  the  visual  purple  separates  as  a  violet  mass.  Under  all  circumstances, 
even  when  still  in  the  retina,  the  visual  purple  is  quickly  bleached  by  direct 
sunlight,  and  with  diffused  light  with  a  rapidity  corresponding  to  the  in- 
tensity of  the  light.  It  passes  from  red  and  orange  to  yellow.  Red  light 
bleaches  the  visual  purple  slowly;  the  ultra-red  light  does  not  bleach  it  at 
all.  A  solution  of  visual  purple  shows  no  special  absorption-bands,  but 
only  a  general  absorption  which  extends  from  the  red  side,  beginning  at 
D  and  extending  to  the  G  line.     The  strongest  absorption  is  found  at  E. 

KoETTGEN  and  Abelsdorf  ^  have  shown  that  there  are,  in  accordance  with 
Kijhne' s  views,  two  varieties  of  visual  purjDle,  the  one  occurring  in  mammals, 
birds,  and  amphibians,  and  the  other,  which  is  more  violet-red,  in  fishes.  The 
first  has  its  maximum  absorption  in  the  green  and  the  other  in  the  yellowish 
green. 

Visual  purple  when  heated  to  52-53°  C.  is  destroyed  after  several  hours, 
and  almost  instantly  when  heated  to  76°  C.  It  is  also  destroyed  by 
alkalies,  acids,  alcohol,  ether,  and  chloroform.  On  the  contrary,  it  resists 
the  action  of  ammonia  or  alum  solution. 

As  the  visual  purple  is  easily  destroyed  by  light,  it  must  therefore  also 
be  regenerated  during  life.  KtJHNE  has  also  found  that  the  retina  of  the 
eye  of  the  frog  becomes  bleached  when  exposed  for  a  long  time  to  strong 
sunUght,  and  that  its  color  gradually  returns  when  the  animal  is  placed  in 
the  dark.  This  regeneration  of  the  visual  purple  is  a  function  of  the  living 
cells  in  the  layer  of  the  pigment-epithelium  of  the  retina.  This  may  be 
inferred  from  the  fact  that  a  detached  piece  of  the  retina  which  has  been 
bleached  by  light  may  have  its  visual  purple  restored  if  it  is  carefully  laid 
on  the  choroid  having  layers  of  the  pigment-epithelium  attached.  The 
regeneration  has,  it  seems,  nothing  to  do  with  the  dark  pigment,  the 
melanin  or  fuscin,  in  the  epithelium-cells.  A  partial  regeneration  seems, 
according  to  Kijhne,  to  be  possible  in  the  retina  which  has  been  completely 
removed.     On  account  of  this  property  of  the  visual  purple  of  being  bleached 

'  Centralbl.  f.  Physiol.,  9;  also  Maly's  Jahresber.,  25,  351. 


VISUAL   PURPLE  AND   VITREOUS  HUMOR.  491 

by  light  during  life  we  may,  as  Kuhxe  has  shown,  under  special  conditions 
and  by  obser\-ing  special  precautions,  obtain  after  death,  by  the  action  of 
intense  light  or  more  continuous  Ught,  the  picture  of  bright  objects,  such 
as  windows  and  the  like — so-called  optograms. 

The  physiological  importance  of  visual  purple  is  unknown.  It  follows 
that  the  visual  purple  is  not  essential  to  sight,  since  it  is  absent  in  certain 
animals  and  also  in  the  cones. 

Visual  purple  must  always  be  prepared  exclusively  in  a  sodium  light. 
It  is  extracted  from  the  net  membrane  by  means  of  a  watery  solution  of 
crystallized  bile.  The  filtered  solution  is  evaporated  in  vacuo  or  dialyzed 
until  the  visual  purple  is  separated.  To  prepare  a  visual-purple  solution 
perfectly  free  from  hsemoglobm  the  solution  of  \'isual  purple  in  cholates  is 
precipitated  by  saturating  with  magnesium  sulphate,  washing  the  precipi- 
tate ^^ith  a  saturated  solution  of  magnesium  sulphate,  and  then  dissohdng 
in  water  by  the  aid  of  the  cholates  simultaneously  precipitated.^ 

The  Pigments  of  the  Cones.  In  the  inner  segments  of  the  cones  of  birds,  rep- 
tiles, and  fishes  a  small  fat-globule  of  var\ang  color  is  found.  Kijhne  ■  has 
isolated  from  this  fat  a  green,  a  yellow,  and  a  red  pigment  called  respectively 
chlorophan,  xanthophan,  and  rhodophan. 

The  dark  pigment  of  the  epithelium-cells  of  the  net  membrane,  which  was 
formerly  called  melanin,  but  has  since  been  named  fuscin  by  Kxjhxe  and  Mays,^ 
contains  iron,  dissolves  in  concentrated  caustic  alkalies  or  concentrated  sulphuric 
acid  on  warming,  but,  like  the  melanins  in  general,  has  been  little  studied.  The 
pigment  occurring  in  the  pigment-cells  of  the  choroid  will  be  discussed  with  the 
melanins  in  Chapter  XVI. 

The  vitreous  humor  is  often  considered  as  a  variety  of  gelatinous 
tissue.  The  membrane  consists,  according  to  C.  Mokxer,  of  a  gelatme- 
formmg  substance.  The  fluid  contains  a  httle  proteid  and  a  mucoid, 
hyalomucoid,  which  was  first  shown  by  Morxer,  and  which  is  precipitated 
by  acetic  acid.  This  contains  12.27  per  cent  N  and  1.19  per  cent  S.  Among 
the  extractives  we  find  a  httle  urea — according  to  Pic.ajid  5  p.  m.,  according 
to  Rahl^l\xx  0.64  p.  m.  Pautz  "*  found  besides  some  urea  paralactic 
acid,  and,  in  confirmation  of  the  statements  of  Chabbas,  Jesxer,  and  Kuhx, 
also  glucose  in  the  vitreous  humor  of  oxen.  The  reaction  of  the  vitreous 
humor  is  alkaline,  and  the  quantity  of  sohds  amoimts  to  about  9-11  p.  m. 
The  quantity  of  mineral  bodies  is  about  6-9  p.  m.  and  the  proteins  0.7  p.  m. 
In  regard  to  the  aqueous  humor  see  page  264. 

'  Kiihne,  Zeitschr.  f.  Biologie,  32. 

^  Kiihne,  Die  nichtbestandigen  Farben  der  Netzhaut,  Untersuch.  aus  dem  physiol. 
Institut  Heidelberg,  1 ,  341. 

ns^uhne,  ibil.,  2,  324. 

*M6mer,  Zeitschr.  f.  physiol.  Chem.,  IS;  Picard,  cited  from  Gamgee,  Physiol. 
Chem.,  1,  454;  Rahlmann,  Maly's  Jahrcsber.,  G;  Pautz,  Zeitschr.  f.  Biologie,  31.  A 
complete  review  of  the  literature  wQl  also  be  found  here. 


492  BRAIN  AND   NERVES. 

The  Crystalline  Lens.  That  substance  which  forms  the  capsule  of  the 
lens  has  been  investigated  by  C.  Morner.  It  belongs,  according  to  him, 
to  a  special  group  of  proteins,  called  memhranins.  The  membranin  bodies 
are  insoluble  at  the  ordinary  temperature  in  water,  salt  solutions,  dilute 
acids,  and  alkalies,  and,  like  the  mucins,  yield  a  reducing  substance  on 
boiling  with  dilute  mineral  acids.  They  contain  lead-blackening  sulphur. 
The  membranins  are  colored  a  very  beautiful  red  by  Millon's  reagent,  but 
give  no  characteristic  reaction  with  concentrated  hydrochloric  acid  or 
Adamkiewicz's  reagent.  They  are  dissolved  with  great  difficulty  by 
pepsin-hydrochloric  acid  or  trypsin  solution,  but  are  soluble  in  dilute  acids 
and  alkalies  in  the  warmth.  Membranin  of  the  capsule  of  the  lens  contains 
14.10  per  cent  N  and  0.83  per  cent  S,  and  is  a  little  less  soluble  than  that 
from  Descemet's  membrane. 

The  chief  mass  of  the  solids  of  the  crystalline  lens  consists  of  proteins, 
whose  nature  has  been  investigated  by  C.  Morner.^  Some  of  these  pro- 
teins dissolve  in  dilute  salt  solution,  while  others  remain  insoluble  in  this 
solvent. 

The  Insoluble  Protein.  The  lens-fibres  consist  of  a  protein  substance 
which  is  insoluble  in  water  and  in  salt  solution  and  to  which  Morner  has 
given  the  name  albumoid.  It  dissolves  readily  in  very  dilute  acids  or 
alkalies.  Its  solution  in  caustic  potash  of  0.1  per  cent  is  very  similar  to  an 
alkali-albuminate  solution,  but  coagulates  at  about  50°  C.  on  nearly  com- 
plete neutralization  and  the  addition  of  8  per  cent  NaCl.  Albumoid  has 
the  following  composition:  C  53.12,  H  6.8,  N  16.62,  and  S  0.79  per  cent. 
The  lens-fibres  themselves  contain  16.61  per  cent  N  and  0.77  per  cent  S. 
The  inner  parts  of  the  lens  are  considerably  richer  in  albumoid  than  the 
outer.  The  quantity  of  albumoid  in  the  entire  lens  amounts  on  an  average 
to  about  48  per  cent  of  the  total  weight  of  the  proteins  of  the  lens. 

The  Soluble  Protein  consists,  exclusive  of  a  very  small  quantity  of  albu- 
min, of  two  globulins,  a-  and  (i-crystallin.  These  two  globuUns  differ  from 
each  other  in  this  manner:  a-crystallin  contains  16.68  per  cent  N  and  0.56 
per  cent  S;  /?-crystallin,  on  the  contrary,  17.04  per  cent  N  and  1.27  per 
cent  S.  The  first  coagulates  at  about  72°  C.  and  the  other  at  63°  C.  Besides 
this,  ^-crystallin  is  precipitated  from  a  salt-free  solution  with  greater  diffi- 
culty and  less  completely  by  acetic  acid  or  carbon  dioxide.  These  globu- 
lins are  not  precipitated  by  an  excess  of  NaCl  at  either  the  ordinary  tem- 
perature or  30°  C.  Magnesium  or  sodium  sulphate  in  substance  precipi- 
tates both  globulins,  on  the  contrary,  at  30°  C.  These  two  globulins  are 
not  equally  divided  in  the  mass  of  the  lens.  The  quantity  of  a-crystallin 
diminishes  in  the  lens  from  without  inwards;  /?-crystallin,  on  the  contrary, 
from  within  outwards. 

Zeitschr.  f.  physiol.  Chem.,  18.     This  contains  also  the  pertinent  literature. 


CRYSTALLINE  LENS.  493 

A.  Bechamp  distinguishes  the  two  following  protein  bodies  in  the  watery- 
extract  of  the  crystalline  lens:  phacozymase,  which  coagulates  at  55°  C,  con- 
tains a  diastatic  enzyme,  and  has  a  specific  rotatory  power  of  («);'=— 41°, 
and  the  crystalbumin,  with  a  specific  rotatory  power  of  («)/=— 80.3°.  From 
the  residue  of  the  lens,  which  was  insoluble  in  water,  Bechamp  extracted,  by 
means  of  hydrochloric  acid,  a  protein  body  having  a  specific  rotatory  power  of 
(a)y=— 80.2°,  which  he  called  crystalfibrin. 

The  lens  does  not  seem  to  contain  any  protein  bodies  which  coagulate 
spontaneously  like  fibrinogen.  That  cloudiness  which  appears  after  death 
depends,  according  to  KIjhne,  upon  the  unequal  changing  of  the  concen- 
tration of  the  contents  of  the  lens-tubes.  This  change  is  produced  by  the 
altered  ratio  of  diffusion.  A  cloudiness  of  the  lens  may  also  be  produced  in 
life  by  a  rapid  removal  of  water,  as,  for  example,  when  a  frog  is  plunged 
into  a  salt  or  sugar  solution.  The  appearance  of  cloudiness  in  diabetes  has 
been  attributed  by  some  to  the  removal  of  water.  The  views  on  this  sub- 
ject are,  however,  contradictory. 

The  average  results  of  four  analyses  made  by  Laptschinsky  ^  of  the 
lens  of  oxen  are  here  given,  calculated  in  parts  per  1000 : 

Proteins 349 . 3 

Lecithin 2.3 

Cholesterin 2.2 

Fat 2.9 

Soluble  salts 5.3 

Insoluble  salts 2.3 

In  cataract  the  amount  of  proteins  is  diminished  and  the  amount  of 
cholesterin  increased. 

The  quantity  of  the  different  proteins  in  the  fresh  moist  lens  of  oxen  is 
as  follows,  according  to  Morner^: 

Albumoid  (lens-fibres) 170  p.  m. 

/?-Crystallin 110  " 

(v-Crystallin 68  " 

Albumin 2  " 

The  corneal  tissue  has  been  previously  considered  (page  434).  The 
sclerotic  has  not  been  closely  investigated,  and  the  choroid  coat  is  chiefly  of 
interest  because  of  the  coloring-matter  (melanin)  it  contains  (see  Chapter 
XVI). 

Tears  consist  of  a  water-clear,  alkaline  fluid  of  a  salty  taste.  Accord- 
ing to  the  analyses  of  Lerch  ^  they  contain  982  p.  m.  water,  18  p.  m.  solids 
with  5  p.  m.  albumin  and  13  p.  m.  NaCl. 

'  Pfliiger's  Arch.,  13. 
M.  c. 

'  Cited  from  v.  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4.  Aufl.,  401. 


494  BRAIN  AND  NERVES. 


THE  FLUIDS  OF  THE  INNER  EAR. 

The  perilymph  and  endolymph  are  alkaline  fluids  which,  besides  salts, 
contain — in  the  same  amounts  as  in  transudates — traces  of  protein,  and  in 
certain  animals  (codfish)  also  mucin.  The  quantity  of  mucin  is  greater  in 
the  perilymph  than  in  the  endolymph. 

Otoliths  contain  745-795  p.  m.  inorganic  substance,  which  consists 
chiefly  of  crystallized  calcium  carbonate.  The  organic  substance  i;:  very 
similar  to  mucin. 


CHAPTER  XIII. 
ORGANS  OF  GENERATION. 

(a)  Male  Generative  Secretions. 

The  testes  have  been  little  investigated  chemically.  We  find  in  the 
testes  of  animals  protein  bodies  of  different  kinds — seralbumin,  alkali  albu- 
minate (?),  and  an  albuminous  body  related  to  Rovida's  hyaline  substance; 
also  leucine,  tyrosine,  creatine,  xanthine  bodies,  cholcsterin,  lecithin,  inosite, 
and  fat.  In  regard  to  the  occurrence  of  glycogen  the  statements  are  some- 
what contradictor}-.  Dareste  ^  found  in  the  testes  of  birds  starch-like 
granules,  which  were  colored  blue  with  difficulty  by  iodine. 

In  the  autolysis  of  the  testes  Levene  ^  found  tvTosine,  alanine,  leucine, 
aminovalerianic  acid,  aminobutj-ric  acid,  a-proline,  phenylalanine,  aspartic  acid, 
glutamic  acid,  and  hypoxanthine.  Pyrimidine  and  hexone  bases  could  not  be 
detected. 

The  semen  as  ejected  is  a  white  or  whitish-yellow,  viscous,  sticky  fluid 
of  a  milky  appearance,  with  whitish,  non-transparent  lumps.  The  milky 
appearance  is  due  to  spermatozoa.  Semen  is  heavier  than  water,  contains 
protei -s,  has  a  neutral  or  faintly  alkaline  reaction  and  a  peculiar  specific 
odor.  Soon  after  ejection  semen  becomes  gelatinous,  as  if  it  were  coagu- 
lated, but  afterwards  becomes  more  fluid.  "VMien  diluted  with  water  white 
flakes  or  shreds  separate  (Henle's  fibrin).  According  to  the  anah'ses  of 
Slow-tzoff,3  human  semen  contains  on  an  average  96. S  p.  m.  solids  with 
9  p.  m.  inorganic  and  87.8  p.  m.  organic  substance.  The  amount  of  pro- 
tein substances  was,  on  an  average,  22.6  p.  m.  and  1.69  p.  m.  of  bodies  solu- 
ble in  ether.  The  protein  substances  consist  of  nucleoproteids,  traces  of 
mucin,  albumin,  and  a  substance  similar  to  proteose  (found  earlier  by 
Posner).  According  to  Cavazzaxi  ^  semen  contains  relatively  considerable 
nucleon,  more  than  any  organ.  The  mineral  bodies  consist  chiefly  of 
calcium  phosphate  and  considerable  NaCl.  Potassium  occurs  only  in 
smaller  amounts. 

'  Compt.  rend.,  7-t. 

*  Amer.  Journ.  of  Physiol.,  11. 

'  Zeiti?chr.  f.  physiol.  Chem.,  35. 

*  Po.sner,  Berl.  klin.  Wochenschr.,  1888,  No.  21,  and  Centralbl.  f.  d.  med.  Wissensch., 
1890;  Cavazzani,  Biochem.  Centralbl.,  1,  o02,  and  Centralbl.  f.  Physiol.,  19. 

495 


496  ORGANS  OF  GENERATION. 

The  semen  in  the  vas  deferens  differs  chiefly  from  the  ejected  semen  in 
that  it  is  without  the  pecuUar  odor.  This  last  depends  on  the  admixture 
•with  the  secretion  of  the  prostate.  This  secretion,  according  to  Iversen, 
has  a  milky  appearance  and  ordinarily  an  alkaline  reaction,  very  rarely  a 
neutral  one,  and  contains  small  amounts  of  proteins,  especially  nucleopro- 
teids,  besides  a  substance  similar  to  fibrinogen  and  to  mucin  (Stern  ^),  and 
mineral  bodies,  especially  NaCl.  Besides  this  it  contains  an  enzyme  vesic- 
ulase  (see  below),  lecithin,  choline  (Stern),  and  a  crystalline  combination 
of  phosphoric  acid  with  a  base,  C2H5N.  This  combination  has  been  called 
Bottcher's  spermine  crystals,  and  it  is  claimed  that  the  specific  odor  of  the 
semen  is  due  to  a  partial  decomposition  of  these  crystals. 

The  crystals  which  appear  on  slowly  evaporating  the  semen,  and  which 
are  also  observed  in  anatomical  preparations  kept  in  alcohol,  are  not  iden- 
tical with  the  Charcot-Leyden  crystals  found  in  the  blood  and  in  the 
lymphatic  glands  in  leucaemia  (Th.  Cohn,  B.  Lewy  2).  They  are,  according 
to  Schreiner,^  as  above  stated,  a  combination  of  phosphoric  acid  with  a 
base,  spermine,  C2H5N,  which  he  discovered. 

Spermine.  The  views  in  regard  to  the  nature  of  this  base  are  not  unanimous. 
According  to  the  investigations  of  Ladenburg  and  Abel,  it  is  not  improbable 
that  spermine  is  identical  with  ethyleneimine ;  but  this  identity  is  disputed  by 
Majert  and  A.  Schmidt,  and  also  by  Poehl.  The  compound  of  spermine  with 
phosphoric  acid — Bottcher's  spermine  crystals — is  insoluble  in  alcohol,  ether, 
and  chloroform,  soluble  with  difficulty  in  cold  water,  but  more  readily  in  hot 
water,  and  easily  soluble  in  dilute  acids  or  alkalies,  also  alkali  carbonates  and 
ammonia.  The  base  is  precipitated  by  tannic  acid,  mercuric  chloride,  gold  chlo- 
ride, platinic  chloride,  potassium-bismuth  iodide,  and  phosphotungstic  acid. 
Spermine  has  a  tonic  action,  and  according  to  Poehl  *  it  has  a  marked  action  on 
the  oxidation  processes  of  the  animal  body. 

On  the  addition  of  a  solution  of  potassium  iodide  and  iodine  to  spermatozoa, 
characteristic  dark-brown  or  bluish-black  crystals  are  obtained — Florence's 
sperm  reaction,  which  is  considered  by  many  as  a  reaction  for  spermine. 
According  to  Bocarius,^  this  reaction  is  due  to  choline. 

Camus  and  Gley^  have  found  that  the  prostate  fluid  in  certain  rodents 
has  the  property  of  coagulating  the  contents  of  the  seminal  vesicles.  This  prop- 
erty is  due  to  a  special  ferment  substance  {vesiculase)  of  the  prostate  fluid. 

'Iversen,  Nord.  med.  Ark.,  C;  also  Maly's  Jahresber.,  4,  358;  Stern,  Biochem. 
Ccntralbl.,  1,748. 

2  Th.  Cohn,  Centralbl.  f.  allg.  Path.  u.  path.  Anat.,  10  (1899);  B.  Lewy,  Centralbl. 
f.  d.  med.  Wis.sen.sch.,  1899,  479. 

3  Annal.  d.  Chem.  u.  Pharm.,  194. 

*  Ladenburg  and  Abel,  Ber.  d.  deutsch.  chem.  Ge.sellsch.,  21;  Majert  and  A.  Schmidt, 
ibid.,  24;  Poehl,  Compt.  rend.,  115,  Berlin,  kliri.  Wochenschr.,  1891  and  1893,  Deutsch. 
med.  Wochenschr.,  1892  and  1895,  and  Zeitschr.  f.  klin.  Med.,  1894. 

^  In  regard  to  Florence's  sperm  reaction,  see  Posner,  Berl.  klin.  Wochenschr. 
1897,  and  Richtcr,  Wien.  klin.  Wochenschr.,  1897;  Bocarius,  Zeitschr.  f.  physiol. 
Chem.,  34. 

•Compt.  rend,  de  see.  biolog.,  48,  49. 


SPERMATOZOA.  497 

The  spermatozoa  show  a  great  resistance  to  chemical  reagents  in  general. 
They  do  not  dissolve  completely  in  concentrated  sulphuric  acid,  nitric  acid, 
acetic  acid,  nor  in  boiling-hot  soda  solutions.  They  are  soluble  in  a  boiling- 
hot  caustic-potash  solution.  They  resist  putrefaction,  and  after  drj-ing  they 
may  be  obtained  again  in  their  original  form  by  moistening  them  with  a  1 
per  cent  common -salt  solution.  By  careful  heating  and  burning  to  an  ash 
the  shape  of  the  spermatozoa  may  be  seen  in  the  ash.  The  quantity  of 
ash  is  about  50  p.  m.  and  consists  mainly  (|)  of  potassium  phosphate. 

The  spermatozoa  show  well-knoviii  movements,  but  the  cause  of  this  is 
not  known.  These  movements  may  continue  for  a  very  long  time,  as  under 
some  conditions  they  may  be  observed  for  several  days  in  the  body  after 
death,  and  in  the  secretion  of  the  uterus  longer  than  a  week.  Acid  Hquids 
stop  these  movements  immediately;  they  are  also  destroyed  by  strong 
alkalies,  especially  ammoniacal  liquids,  also  by  distilled  water,  alcohol, 
ether,  etc.  The  movements  continue  for  a  longer  time  in  faintly  alkaline 
liquids,  especially  in  alkaline  animal  secretions,  and  also  in  properly 
diluted  neutral  salt  solutions. 

Spermatozoa  are  nucleus  formations  and  hence  are  rich  in  nucleic  acid, 
which  exists  in  the  heads.  The  tails  contain  protein  and  are  besides  this 
rich  in  lecithin,  cholesterin,  and  fat,  which  bodies  occur  only  to  a  small 
extent  (if  at  all)  in  the  heads.  The  tails  seem  by  their  composition  to  be 
closely  allied  to  the  non-meduUated  nerv^es  or  the  axis-cylinders.  In  the 
various  kinds  of  animals  investigated,  the  head  contains  nucleic  acid,  which 
in  fishes  is  partly  combined  ^vith  protamines  and  partly  wdth  histones.  In 
other  animals,  such  as  the  bull  and  boar,  protein-like  substances  occur  with 
the  nucleic  acid,  but  no  protamine. 

Our  knowledge  of  the  chemical  composition  of  spermatozoa  has  been 
greatly  enhanced  by  the  important  investigations  of  Miescher  i  on  salmon 
milt.  The  intermediate  fluid  of  the  spermatozoa  of  Rhine  salmon  is  a 
dilute  salt  solution  containing  1.3-1.9  p.  m.  organic  and  6.5-7.5  p.  m. 
inorganic  bodies.  The  last  consist  chiefly  of  sodium  chloride  and  carbonate, 
besides  some  potassium  chloride  and  sulphate.  The  fluid  contains  only 
traces  of  protein,  but  no  peptone.  The  tails  consist  of  419  p.  m.  protein, 
318.3  p.  m.  lecithin,  and  262.7  p.  m.  cholesterin  and  fat.  The  heads 
extracted  '^ith  alcohol-ether  contain  on  an  average  960  p.  m.  protamine 
nucleate,  which  nevertheless  is  not  uniform,  but  is  so  di\dded  that  the  outer 
layers  consist  of  basic  protamine  nucleate,  while  the  inner  layers,  on  the 
contrary,  consist  of  acid  protamine  nucleate.  Besides  the  protamine 
nucleate  there  are  present  in  the  heads,  although  to  a  very  slight  extent 
unkno^\Tl    organic    substances.     The    unrij^e    salmon    spermatozoa,    while 


'See  Miescher,  "Die  histochemischen  und  physiologischen  Arbeiten  von  Friedrich 
Miescher,  gesammclt  und  herausgegeben  von  seinen  Freunden,"  Leipzig,  1897. 


498  ORGANS  OF  GENERATION. 

developing,  also  contain  nucleic  acid,  but  no  protamine,  with  a  protein 
substance,  "  alhuminose,"  which  probably  is  a  step  in  the  formation  of 
protamine.  According  to  Kossel  and  Mathews,^  in  the  herring  as  in 
the  salmon,  the  heads  of  the  spermatozoa  consist  of  protamine  nucleate 
but  no  free  protein. 

Spennatin  is  a  name  which  has  been  given  to  a  constituent  similar  to  alkali 
albuminate,  but  it  has  not  been  closely  studied. 

Prostatic  concrements  are  of  two  kinds.  One  is  very  small,  generally  oval  in 
shape,  with  concentric  layers.  In  young  but  not  in  older  persons  they  are  colored 
blue  by  iodine  (Iversen  ')•  The  other  kind  is  larger,  sometimes  the  size  of  the 
head  of  a  pin,  and  consisting  chiefly  of  calcium  phosphate  (about  700  p.  m.),  with 
only  a  very  small  amount  (about  160  p.  m.)  of  organic  substance. 

(b)  Female  Generative  Organs. 

The  stroma  of  the  ovaries  is  of  little  interest  from  a  physiologico- 
chemical  standpoint,  and  the  most  important  constituents  of  the  ovaries, 
the  Graafian  follicles  with  the  ovum,  have  not  thus  far  been  the  subject 
of  a  careful  chemical  investigation.  The  fluid  in  the  follicles  (of  the  cow) 
does  not  contain,  as  has  been  stated,  the  peculiar  bodies,  paralbumin  or 
metalbumin,  which  are  found  in  certain  pathological  ovarial  fluids,  but 
seems  to  be  a  serous  liciuid.  The  corpora  lutea  are  colored  yellow  by  an 
amorphous  pigment  called  lutein.  Besides  this  another  coloring-matter 
sometimes  occurs  which  is  not  soluble  in  alkali;  it  is  crystalUne,  but  not 
identical  with  bilirubin  or  ha?matoidin;  but  it  may  be  identified  as  a  lutein 
by  its  spectroscopic  behavior  (Piccolo  and  Lieben,  KiJHNE  and  Ewald  ■*). 

The  cysts  often  occurring  in  the  ovaries  are  of  special  pathological 
interest,  and  these  may  have  essentially  different  contents,  depending 
upon  their  variety  and  origin. 

The  serous  cysts  (Hydrops  folliculorum  Graafii),  which  are  formed 
by  a  dilation  of  the  Graafian  follicles,  contain  a  serous  liquid  which  has  a 
specific  gravity  of  1.(305-1.022.  A  specific  gravity  of  1.020  is  less  frequent. 
Generally  the  specific  gravity  is  lower,  1.005-1.014,  with  10-40  p.  m.  solids. 
As  far  as  is  known,  the  contents  of  these  cysts  do  not  essentially  differ  from 
other  serous  liquids. 

The  proliferous  cysts  (myxoid  cysts,  colloid  cysts),  which  are  devel- 
oped from  Pfluger's  epithelium-tubes,  may  have  a  content  of  a  decidedly 
variable  composition. 

We  sometimes  find  in  small  cysts  a  semi-solid,  transparent,  or  somewhat 
cloudy  or  opalescent  mass  which  appears  like  solidified  glue  or  quivering 
jelly,  and  which  has  been  called  colloid  because  of  its  physical  properties. 
In  other  cases  the  cysts  contain  a  thick,  tough  mass  which  can  be  drawn  out 

»  Zeitschr.  f.  physiol.  Cheni.,  23.         '  Nord.  med.  Ark.,  6.         '  See  Chapter  VI,  p.  216. 


COLLOID.  499 

into  long  threads,  and  as  this  mass  hi  the  different  cysts  is  more  or  less 
diluted  mth  serous  liquids  their  contents  may  have  a  variable  consistency. 
In  still  other  cases  the  small  cysts  may  also  contain  a  thin,  watery  fluid. 
The  cobr  of  the  contents  is  also  variable.  Sometimes  they  are  bluish 
white,  opalescent,  and  again  they  are  yellow,  yellowish  browii,  or  yellowish 
with  a  shade  of  green.  They  are  often  colored  more  or  less  chocolate- 
brown  or  red-brown,  due  to  the  decomposed  blood-coloring  matters.  The 
reaction  is  alkaline  or  nearly  neutral.  The  specific  gravity,  which  may 
vary  considerably,  is  generally  1.015-1.030,  but  may  occasionally  be  1.005- 
1.010  or  1.050-1.055.  The  amount  of  solids  is  very  variable.  In  rare 
cases  it  amounts  to  only  10-20  p.  m.;  ordinarily  it  varies  between  50-70- 
100  p.  m.     In  a  few  instances  150-200  p.  m.  solids  have  been  found. 

As  form-elements  one  finds  red  and  white  blood-corpuscles,  granular 
cells,  partly  fat-degenerated  epithelium  and  partly  large  so-called  Gluge's 
corpuscles,  fine  granular  masses,  epitheliuin-cells,  cholesterin  crystals,  and 
colloid  corpuscles — large,  circular,  highly  refractive  formations. 

Though  the  contents  of  the  proliferous  cyst  may  have  a  variable  compo- 
sition, still  it  may  be  characterized  in  typical  cases  by  its  slimy  or  ropy 
consistency;  by  its  grayish-yellow,  chocolate-browTi,  sometimes  whitish- 
gray  color;  and  by  its  relatively  high  specific  gravity,  1.015-1.025.  Such  a 
liquid  does  not  ordinarily  show  a  spontaneous  fibrin  coagulation. 

We  consider  colloid,  metalhumin,  and  paralbumin  as  characteristic  con- 
stituents of  these  cysts. 

Colloid.  This  name  does  not  designate  any  particular  chemical  sub- 
stance, but  is  given  to  the  contents  of  tumors  with  certain  physical  proper- 
ties similar  to  gelatine  jelly.  Colloid  is  found  as  a  pathological  product 
in  several  organs. 

Colloid  is  a  gelatinous  mass,  insoluble  in  water  and  acetic  acid;  it  is 
dissolved  by  alkalies  and  gives  a  liquid  which  is  not  precipitated  by  acetic 
acid  or  by  acetic  acid  and  potassium  ferrocyanide.  According  to  Pfannen- 
STiEL  1  such  a  colloid  is  designated  /?-pseudomucin.  Sometimes  a  colloid  is 
found  which,  when  treated  with  a  verj'  dilute  alkali,  gives  a  solution  similar 
to  a  mucin  solution.  Colloid  is  very  closely  related  to  mucin  and  is  con- 
sidered by  certain  investigators  as  a  modified  mucin.  An  ovarial  colloid 
analyzed  by  Panzer  contained  931  p.  m.  water,  57  p.  m.  organic  substance, 
and  12  p.  m.  ash.  The  elementary  composition  w^as  C  47.27,  H  5.86,  N 
8.40,  S  0.79,  P  0.54,  and  ash  6.43  per  cent.  A  colloid  found  by  Wurtz  2 
in  the  lungs  contained  C  48.09,  H  7.47,  N  7.00,  and  0(  +  S)  37.44  per  cent. 
Colloids  of  different  origin  seem  to  be  of  varsing  composition. 


»Aich.  f.  Gynak.,38. 

'Panzer,  Zeitschr.   f.  physiol.  Chem.,  28;   Wurtz,  see  Lebert,  Beitr.  zur  Kenntnis 
des  Gallertkrebses,  Virchow's  Arch.,  4. 


500  ORGANS   OF  GENERATION. 

Metalhumin.  This  name  Scherer  ^  gave  to  a  protein  substance  found 
by  him  in  an  ovarial  fluid.  The  metalbumin  was  considered  by  Scherer 
to  be  an  albuminous  body,  but  it  belongs  to  the  mucin  group,  and  it  is  for 
this  reason  called  pseudomucin  by  Hammarsten.^ 

Pseudomucin.  This  body,  which,  like  the  mucms,  gives  a  reducing 
substance  when  boiled  with  acids,  is  a  mucoid  of  the  following  composition: 
C  49.75,  H  6.98,  N  10.28,  S  1.25,  0  31.74  per  cent  (.Hammarsten).  With 
water  pseudomucin  gives  a  slimy,  ropy  solution,  and  it  is  this  substance 
which  gives  the  fluid  contents  of  the  ovarial  cysts  their  typical  ropy  prop- 
erty. Its  solutions  do  not  coagulate  on  boiling,  but  only  become  milky 
or  opalescent.  Unlike  mucin,  pseudomucin  solutions  are  not  precipitated 
by  acetic  acid.  With  alcohol  they  give  a  coarse  flocculent  or  thready 
precipitate  which  is  soluble  even  after  having  been  kept  under  water  or 
alcohol  for  a  long  time. 

Paralbumin  is  another  substance  discovered  by  Scherer,  which  occurs 
in  ovarial  liquids  and  also  in  ascitic  fluids  with  the  simultaneous  presence 
of  ovarial  cysts  and  rupture  of  the  same.  It  is  therefore  only  a  mixture 
of  pseudomucin  with  variable  amounts  of  protein,  and  the  reactions  of 
paralbumin  are  correspondingly  variable. 

MiTJUKOFF  ^  has  isolated  and  investigated  a  colloid  from  an  ovarial  cyst.  It 
had  the  following  composition:  C  51.76,  H  7.76,  N  10.7,  S  1.09,  and  O  28.69  per 
cent,  and  differed  from  mucin  and  pseudomucin  by  reducing  Fehling's  solution 
before  boiling  with  acid.  It  must  be  remarked  that  pseudomucin,  on  boiling 
sufficiently  long  with  alkali,  or  by  the  use  of  a  concentrated  solution  of  caustic 
alkali,  also  splits  and  causes  a  reduction.  This  reduction  is  nevertheless  weak 
as  compared  with  that  produced  after  boiling  with  an  acid.  The  body  isolated 
by  MiTJUKOFF  is  called  paramudii. 

The  pseudomucin  as  well  as  colloid  are  mucoid  substances,  and  the 
carbohydrate  obtained  from  them  is  glucosamine  (chitosamine),  as  espe- 
cially shown  by  Fr.  Muller,  Neuberg  and  Heymann."*  From  pseudo- 
mucin Zangerle  ^  obtained  30  per  cent  glucosamine,  and  Neuberg  and 
Heymann  have  shown  that  the  glucosamine  is  the  only  carbohydrate 
regularly  taking  part  in  the  structure  of  these  substances.  Still  there  are 
also  statements  as  to  the  occurrence  of  chondroitin-sulphuric  acid  (or  an 
allied  acid)  in  pseudomucin  or  colloid  (Panzer),  but  this  is  not  constant 
according  to  the  experience  of  Hammarsten. 

»  Verb.  d.  physik.-med.  Gesellsch.  in  Wiirzburg,  2,  and  Sitzungsber.  der  physik.- 
med.  Gesellsch.  in  Wiirzburg  fiir  1864-1865;  Wiirzburg  med.  Zeitschr.,  7,  No.  6. 

^  Zeitsohr.  f.  physiol.  Chem.,  G. 

3K.  Mitjukoff,  Arch.  f.  Gyniikol.,  49. 

"Muller,  Verh.  d.  Naturf.  Gesellsch.  in  Basel,  12,  part  2;  Neuberg  and  Heymann, 
Hofmeister's  Beitrage,  2.     See  also  Leathes,  Arch.  f.  cxp.  Path.  u.  Pharm.,  43. 

*Miinch.  med,  Wochenschr.,  1900. 


PSEUDOMUCIN.  501 

As  hydrolytic  cleavage  products  of  pseud omucin  Otori  ^  has  obtained, 
besides  carbohydrate  derivatives  such  as  levulinic  acid  and  humus  sub- 
stances, leucine,  tyrosine,  glycocoll,  aspartic  acid,  glutamic  acid,  valerianic 
acid,  arginine,  lysine,  and  guanidine.  The  quantity  of  guanidine,  it  seems, 
was  greater  than  that  which  could  be  derived  from  the  arginine,  hence 
this  body  probably  originated  from  another  complex. 

The  detection  of  metalbumin  and  paralbumin  is  naturally  connected 
with  the  detection  of  pseudomucin.  A  typical  ovarial  fluid  containing 
pseudomucin  is,  as  a  rule,  sufficiently  characterized  by  its  physical  proper- 
ties, and  a  special  chemical  investigation  is  necessary  only  in  cases  where  a 
serous  fluid  contains  ven,^  small  amounts  of  pseudomucin.  The  procedure 
is  as  follows:  The  protein  is  removed  by  heating  to  boiling  with  the  addition 
of  acetic  acid;  the  filtrate  is  strongly  concentrated  and  precipitated  by 
alcohol.  The  precipitate,  a  transformation  product  of  pseudomucin, 
is  carefully  washed  wdth  alcohol  and  then  dissolved  in  water.  A  part  of 
this  solution  is  digested  with  saliva  at  the  temperature  of  the  body  and  then 
tested  for  glucose  (derived  from  glycogen  or  dextrin).  If  glycogen  is  pres- 
ent, it  will  be  converted  into  glucose  by  the  saliva;  precipitate  again  with 
alcohol  and  then  proceed  as  in  the  absence  of  glycogen.  In  this  last-men- 
tioned case,  first  add  acetic  acid  to  the  solution  of  the  alcohol  precipitate 
in  water  so  as  to  precipitate  any  existing  mucin.  The  precipitate  produced 
is  filtered  off,  the  filtrate  treated  with  2  per  cent  HCl  and  warmed  on  the 
water-bath  until  the  liquid  is  deep  browii  in  color.  In  the  presence  of 
pseudomucin  this  solution  gives  Trommer's  test. 

The  other  protein  bodies  which  have  been  found  in  cystic  fluids  are 
serglohulin  and  seralbumin,  peptone  (?),  mucin,  and  mucin-peptone  (?). 
Fibrin  occurs  only  in  exceptional  cases.  The  quantity  of  mineral  bodies 
on  an  average  amounts  to  about  10  p.  m.  The  amount  of  extractive 
bodies  (cholesterin  and  urea)  and  fat  is  ordinarily  2-4  p.  m.  The  remaining 
solids,  w^hich  constitute  the  chief  mass,  are  protein  bodies  and  pseudomucin. 

The  intraligamentary,  papillary  cysts  contain  a  yellow,  yellowish- 
green,  or  bro^vnish-green  liquid  which  contains  either  no  pseudomucin  or 
ver}^  little.  The  specific  gravity  is  generally  rather  high,  1.032-1.036, 
with  90-100  p.  m.  solids.  The  principal  constituents  are  the  simple  proteins 
of  blood-serum. 

The  rare  tubo-ovarial  cysts  contain  as  a  rule  a  watery,  serous  fluid  con- 
taining no  pseudomucin. 

The  parovarial  cysts  or  the  cysts  of  the  ligamenta  lata  may  attain  a 
considerable  size.  In  general,  and  when  quite  typical,  the  contents  are 
watery,  mostly  very  pale  yellow-colored,  water-clear  or  only  slightly  opal- 
escent liquids.  The  specific  gravity  is  low,  1.002-1.009,  and  the  solids  only 
amount  to  10-20  p.  m.  Pseudomucin  does  not  occur  as  a  typical  constit- 
uent; protein  is  sometimes  absent,  and  when  it  does  occur  the  quantity  is 

'  Zeitschr.  f.  physiol.  Chem.,  42  and  43. 


502  ORGANS  OF  GENERATION. 

very  small.  The  principal  part  of  the  solids  consists  of  salts  and  extrac- 
tive bodies.  In  exceptional  cases  the  fluid  may  be  rich  in  protein  and  may 
show  a  higher  specific  gravity. 

In  regard  to  the  quantitative  composition  of  the  fluid  from  ovarial  cysts 
we  refer  the  reader  to  the  work  of  Oerum.'^ 

E.  LuDwiG  and  R.  v.  Zeynek  '  have  recently  investigated  the  fat  from 
dermoid  cysts.  Besides  a  little  arachidic  acid,. they  found  oleic,  stearic,  palmitic, 
and  myristic  acids,  cetyl  alcohol,  and  a  cholesterin-like  substance. 

The  colloid  from  a  uterine  fibroma  analyzed  by  Stollmann  ^  contained  a 
pseudomucin  soluble  in  water  and  a  colloid  (paramucin)  insoluble  in  water,  both 
of  which  behaved  differently  with  alcohol  as  compared  with  the  corresponding 
substances  from  ovarial  cysts. 

The  Ovum. 

The  small  ova  of  man  and  mammals  cannot,  for  evident  reasons,  be  the 
subject  of  a  searching  chemical  investigation.  Up  to  the  present  time  the 
eggs  of  birds,  amphibians,  and  fishes  have  been  investigated,  but  above  all 
the  hen's  egg.  We  will  here  occupy  ourselves  with  the  constituents  of  this 
last. 

The  Yolk  of  the  Hen's  Egg.  In  the  so-called  white  yolk,  which  forms 
the  germ  with  a  process  reaching  to  the  centre  of  the  yolk  {latehra),  and  form- 
ing a  layer  between  the  yolk  and  yolk-membrane,  there  occur  'protein, 
nuclein,  lecithin,  and  potassium  (Liebermann  ■*).  The  occurrence  of  gly- 
cogen is  doubtful.  The  yolk-membrane  consists  of  an  albuminoid  similar 
in  certain  respects  to  keratin  (Liebermann). 

The  principal  part  of  the  yolk — the  nutritive  yolk  or  yellow — is  a 
viscous,  non-transparent,  pale-yellow  or  orange-yellow  alkaline  emulsion 
of  a  mild  taste.  The  yolk  contains  vitellin,  lecithin,  cholesterin,  fat,  color- 
ing-matters, traces  of  neuridine  (Brieger'"'),  purine  bases  (Mesernitzki  ^), 
glucose  in  very  small  quantities,  and  mineral  bodies.  The  occurrence  of 
cerebrin  and  of  granules  similar  to  starch  (Dareste  '^)  has  not  been  posi- 
tively proved. 

Several  enzymes  have  teen  found  in  the  yolk,  especially  a  diastatic 
enzyme  (Muller  and  Masuyama),  a  glycolytic  enzyme  (Stepanek)  which 
in  the  absence  of  air  brings  about  an  alcohoUc  fermentation  of  sugar  and 


*  Kemiske  Studier  over  Ovariecystevsedsker,  etc.,  Koebenhavn,  1884.     See  also 
Maly's  Jahresber.,  14,  459. 

2  Zeitschr.  f.  physiol.  Chem.,  23. 

'  American  Gynecology,  March,  1903. 

♦Pfliiger's  Arch.,  43. 

^Ueber  Ptomaine,  Berlin,  1885. 

^Mesernitzki,  Biochem.  Centralbl.,  1,  739. 

'Compt.  rend.,  72. 


OVOVITELLIN.  503 

in  the  presence  of  air  forms  Ccarbon  dioxide  and  lactic  acid,  and  finally  a 
proteolytic  (Wohlgemuth),  a  lipolytic,  and  a  chromolytic  (?)  enzyme. ^ 

Ovovitellin.  This  body,  which  is  generally  considered  as  a  globulin,  is 
in  reality  a  nucleoalbumin.  The  question  as  to  what  relationship  other 
protein  substances  which  are  related  to  ovovitelUn,  like  the  aleuron  grains 
of  certain  seeds  and  the  yolk  spherules  of  the  eggs  of  certain  fishes  and 
amphibians,  bear  to  this  substance  is  one  which  requires  further  investi- 
gation. 

The  ovo\dtellin  which  has  been  prepared  from  the  yolk  of  eggs  is  not  a 
pure  protein  body,  but  always  contains  lecithin.  Hoppe-Seyler  found 
25  per  cent  lecithin  in  vitellin.  The  lecithin  may  be  removed  by  boiling 
alcohol,  but  the  vitellin  is  changed  thereby,  and  it  is  therefore  probable  that 
the  lecithin  is  chemically  united  with  the  vitellin  (Hoppe-Seyler  2).  Ac- 
cording to  Osborne  and  Campbell,  the  so-called  ovovitellin  is  a  mixture 
of  various  vitellin-lecithin  combinations,  with  15-30  per  cent  of  lecithin. 
The  protein  substance  freed  from  lecithin  is  the  same  in  all  these  compounds 
and  has  the  following  composition:  C  51.24,  H  7.16,  N  16.38,  S  1.04,  P  0.94, 
O  23.24  per  cent.  These  figures  differ  somewhat  from  those  obtained  by 
Gross  3  for  vitellin  prepared  by  another  method  (precipitation  with 
(NH4)2SO.i),  namely,  C  4SX)1,  H  6.35,  N  14.91-16.97,  P  0.32-0.35,  S  0.88, 
and  the  composition  of  ovovitellin  is  therefore  not  positively  known. 
Gross  found  in  vitellin  a  globulin  coagulathig  at  76-77°  C.  in  a  solution 
containing  hydrochloric  acid. 

On  the  pepsin  digestion  of  ovovitellin,  Osborne  and  Campbell  obtained 
a  pseud  on  uclein  with  varying  amomits  of  phosphorus,  2.52-4.19  per  cent. 
BuNGE  ^  prepared  a  pseudonuclein  by  digesting  ihe  yolk  witJi  gastric  juice, 
and  his  pseudonuclein,  according  to  him,  is  of  great  importance  m  the 
formation  of  the  blood,  and  on  these  grounds  he  called  it  hcvmatogen.  This 
hsematogen  has  the  following  composition:  C  42.11,  H  6.08,  N  14.73,  S  0.55, 
P  5.19,  Fe'0.29,  and  O  31.05  per  cent.  The  composition  of  this  substance 
may  vary  considerably  even  on  using  the  same  method  of  preparation. 

Vitellin  is  similar  to  the  globulins  in  that  it  is  insoluble  in  water,  but  on 
the  contrary  soluble  in  dilute  neutral-salt  solutions  (although  the  solution 
is  not  quite  transparent").  It  is  also  soluble  in  hydrochloric  acid  of  1  p.  m. 
and  in  very  dilute  solutions  of  alkalies  or  alkali  carbonates.  It  is  precipi- 
tated from  its  salt  solution  by  diluting  with  water,  and  when  allowed  to 

'  MiiUer  and  Masuyama,  Zeitschr.  f.  Biologie,  30;  Stepanek,  Centralbl.  f.  Physiol., 
18, 188;  Wohlgemuth  in  Salkowski's  Festschrift  and  Zeitschr.  f.  physiol.  Chem.,  44. 

'  Med.  chem.  Untersuch.   216. 

'  Osborne  and  Campbell,  Connecticut  Agric.  Exp.  Station,  23d  Ann.  Report,  New 
Haven.  1900;   Gross,  Zur  Ke^mtn.  d.  OvoviteUins.  Inaug.-Diss.  Stra^ssburg,  1899. 

^  Zeitpchr.  f.  physiol.  Chem.,  9,  49.  See  also  Hugo_:nenq  and  Morel,  Compt.  rend., 
140  and  141. 


504  ORGANS  OF  GENERATION. 

stand  some  time  in  contact  with  water  the  vitellin  is  gradually  changed, 
forming  a  substance  more  like  the  albumir  ates.  The  coagulation  tempera- 
ture for  the  solution  containing  salt  (NaCl)  lies  between  70°  and  75°  C, 
or,  when  heated  very  rapidly,  at  about  80°  C.  Vitellin  differs  from  the 
globulins  in  yielding  pseudonuclein  by  peptic  digestion.  It  is  not  always 
completely  precipitated  by  NaCl  in  substance.  The  ovovitellin  isolated  by 
Gross  gave  Molisch's  reaction.  Neuberg  ^  has  also  split  off  glucosamine 
from  the  yolk  and  has  identified  it  as  norisosaccharic  acid.  It  is  difficult 
to  state  whether  this  glucosamine  was  derived  from  the  vitellin  or  from 
some  other  constituent  of  the  yolk. 

The  chief  points  in  the  preparation  of  ovovitellin  are  as  follows:  The 
yolk  is  thoroughly  agitated  with  ether;  the  residue  is  dissolved  in  a  10  per 
cent  common-salt  solution,  filtered,  and  the  vitellin  precipitated  by  adding 
an  abundance  of  water.  The  vitellin  is  now  purified  by  repeatedly  redis- 
solving  in  dilute  common-salt  solutions  and  precipitating  with  water. 

Ichthulin,  which  occurs  in  the  eggs  of  the  carp  and  other  fishes,  is,  according 
to  KossEL  and  Walter,  an  amorphous  modification  of  the  crystalline  body 
ichthidin,  which  occurs  in  the  eggs  of  the  carp.  Ichthulin  is  precipitated  on 
diluting  with  water.  It  used  to  be  considered  as  a  vitelHn.  According  to  Walter 
it  yields  a  pseudonuclein  on  peptic  digestion;  and  this  pseudonuclein  gives  a 
reducing  carbohydrate  on  boiling  with  sulphuric  acid.  Ichthulin  has  the  follow- 
ing composition:  C  53.42,  H  7.63,  N  15.63,  O  22.19,  S  0.41,  P  0.43.  It  also  con- 
tains iron.  The  ichthulin  investigated  by  Levene  from  codfish  eggs  had  the 
composition  C  52.44,  H  7.45,  N  15.96,  S  0.92,  P  0.65,  Fe+O  22.58  per  cent,  and 
yielded  no  reducing  substances  on  boiling  with  acids.  The  pure  vitellin  isolated 
by  Hammarsten^  from  perch  eggs  had  a  similar  behavior  and  was  very  readily 
changed  by  a  little  hydrochloric  acid  so  that  it  was  converted  into  a  typical 
pseudonuclein.  The  codfish  ichthulin  yielded  a  pseudonucleic  acid  with  10.34 
per  cent  phosphorus,  but  this  acid  still  gave  the  protein  reactions. 

The  yolk  also  contains  albumin,  besides  vitellin  and  the  above-mentioned 
globulin. 

The  fat  of  the  yolk  of  the  egg  is,  according  to  Liebermann,  a  mixture 
of  a  solid  and  a  liquid  fat.  The  solid  fat  consists  chiefly  of  tripalmitin  with 
some  tristearin.  On  the  saponification  of  the  egg-oil  Liebermann  obtained 
40  per  cent  oleic  acid,  38.04  per  cent  palmitic  acid,  and  15.21  per  cent  stearic 
acid.  The  fat  of  the  yolk  of  the  egg  contains  less  carbon  than  other  fats, 
which  may  depend  upon  the  presence  of  monoglycerides  and  diglycerides,  or 
upon  a  quantity  of  fatty  acid  deficient  in  carbon  (Liebermann).  In  the 
lecithin,  or  more  correctly  in  the  lecithin  mixture  of  the  yolk,  Cousin  finds 
also  linolic  acid  besides  the  three  ordinary  fatty  acids.  The  composition 
of  yolk  fat  is  dependent  upon  the  food,  as  Henriques  and  Hansen  ^  have 
shown  that  the  fat  of  the  food  passes  into  the  egg. 

'  Ber.  d.  d.  chem.  Gesellsch.,  34. 

nValter,  Zeitschr.  f.  physiol.  Chem.,  15;  Levene,  ibid.,  32;  Hammarsten,  Skand. 
Arch.  f.  Physiol.,  17. 

*  Cousin,  Compt.  rend.,  137;  Henriques  and  Hansen,  Skand.  Arch.  f.  Physiol.,  14. 


LUTEIN.  505 

Lutein.  Yellow  or  orange-red  amorphous  coloring-matters  occur  in  the 
yellow  of  the  egg  and  in  several  other  places  in  the  animal  organism;  for 
instance,  in  the  blood-serum  and  serous  fluids,  fatty  tissues,  milk-fat,  corpora 
lutea,  and  in  the  fat-globules  of  the  retina.  These  coloring-matters,  which 
also  occur  in  the  vegetable  kingdom  (Thudichum),  and  whose  relationship 
to  the  vegetable  pigments,  the  xanthophyll  group,  has  recently  been  shown 
by  ScHUxcK,^  have  been  called  luteins  or  lipochromes. 

The  luteins,  which  among  themselves  show  somewhat  different  proper- 
ties, are  all  soluble  in  alcohol,  ether,  and  chloroform.  They  differ  from  the 
bile-pigment,  bilirubin,  in  that  they  are  not  separated  from  their  solution 
in  chloroform  by  water  containing  alkali,  and  also  in  that  they  do  not  give 
the  characteristic  play  of  colors  with  nitric  acid  containing  a  little  nitrous 
acid,  but  give  a  transient  blue  color,  and,  lastly,  they  ordinarily  show  an 
absorption-spectrum  of  two  bands,  of  which  one  covers  the  line  F  and  the 
other  lies  between  the  lines  F  and  G.  Tlie  luteins  ^^ithstand  the  action  of 
alkalies  so  that  they  are  not  changed  when  we  remove  the  fats  present  by 
means  of  saponification. 

Lutein  has  not  been  prepared  pure.  Maly^  has  found  two  pigments  free  from 
iron  in  the  eggs  of  a  water-spider  (Maja  squinado) — one  a  red  (vitelloriihin)  and 
the  other  a  j-eUow  pigment  {vitellolutein) .  Both  of  these  pigments  are  colored 
blue  by  nitric  acid  containing  nitrous  acid  and  beautifully  green  by  concentrated 
sulphuric  acid.  The  absorption-bands,  especially  of  the  vitellolutein,  correspond 
very  nearly  to  those  of  ovolutein. 

The  mineral  bodies  of  the  yolk  of  the  egg  consist,  according  to  Poleck,^ 
of  51.2-65.7  parts  soda,  80.5-89.3  potash,  122.1-132.8  lime,  20.7-21.1 
magnesia,  11.90-14.5  iron  oxide,  638.1-667.0  phosphoric  acid,  and  5.5-14.0 
parts  silicic  acid  in  1000  parts  of  the  ash.  We  fhid  phosphoric  acid  and 
lime  the  most  abimdant,  and  then  potash,  which  is  somewhat  greater  in 
quantity  than  the  soda.  These  results  are  not,  however,  quite  correct :  first, 
because  no  dissolved  phosphate  occurs  in  the  yolk  (Liebermaxx),  and 
secondly,  in  burning,  phosphoric  and  sulphuric  acids  are  produced,  and  these 
drive  away  the  chlorine,  which  is  not  accotmted  for  in  the  preceding 
analyses. 

The  yolk  of  the  hen's  egg  weighs  about  12-18  grams.  The  quantity 
of  water  and  solids  amounts,  according  to  Paeke,'*  to  471.9  p.  m,  and 
528.1  p.  m.  respectively.  Among  the  solids  he  found  156.3  p.  m.  protein, 
3.53  p.  m.  soluble  and  6.12  p.  m.  insoluble  salts.      The   c^uantity   of   fat, 


•Thudichum,  Centialbl.  f.  d.  med.  Wissensch..  1869;    Schunck,  see  Chem.  Cen- 
tralbl.,  1903,  2,  119.5. 

*  Monatshefte  f.  Chem.,  2. 

^  Cited  from  v.  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4.  Aufl.,  740. 

^  Hoppe-Seyler,  Med.  chem.  L'ntersuch.,  Heft  2,  209. 


506  ORGANS  OF   GENERATION. 

according  to  Parke,  is  228.4  p.  m.;  the  lecithin,  calculated  from  the  amount 
of  phosphorus  in  the  organic  substance  of  the  alcohol -ether  extract,  was 
107.2  p.  m.  and  the  cholesterin  17.5  p.  m. 

The  white  of  the  egg  is  a  faintly  yellow  albuminous  fluid  inclosed  in  a 
framework  of  thin  membranes;  and  this  fluid  is  in  itself  very  liquid,  but 
seems  viscous  because  of  the  presence  of  these  fine  membranes.  That  sub- 
stance which  forms  the  membranes,  and  of  which  the  chalaza  consists,  seems 
to  be  a  body  nearly  related  to  horn  substances  (Liebermann). 

The  white  of  the  egg  has  a  specific  gravity  of  1.045  and  always  has  an 
alkaline  reaction  towards  litmus.  It  contains  850-880  p.  m.  water,  100-130 
p.  m.  protein  bodies,  and  7  p.  m.  salts.  Among  the  extractive  bodies 
Lehmann  found  a  fermentable  variety  of  sugar  which  amounted  to  5  p.  m. 
or,  according  to  Meissner,  80  p.  m.  of  the  solids.^  Besides  these  one  finds 
in  the  white  of  the  egg  traces  of  fats,  soaps,  lecithin  and  cholesterin. 

The  white  of  the  egg  of  the  Insessores  becomes  transparent  on  boiling  and  acts 
in  many  respects  like  alkali  albuminate.  This  albumin  Tarchanoff^  called 
"  tatalbmyiin." 

The  protein  substances  of  the  whit-e  of  egg  are  all  glucoproteids,  as  they 
all  yield  glucosamine.  According  to  the  solution  and  precipitation  prop- 
erties they  are  similar  to  the  globulins,  albumins  or  proteoses.  The  repre- 
sentatives of  the  first  two  groups,  which  until  recently  were  considered 
as  tine  proteins,  are  ovoglohulin  and  ovalbumin.  The  proteose-like  body 
is  ovomucoid. 

Ovoglobulin  separates  in  part  on  diluting  the  egg-white  vnih  water. 
It  is  precipitated  upon  saturation  with  magnesium  sulphate  or  upon 
one-half  saturation  with  ammonium  sulphate  and  coagulates  at  about  75°  C. 
By  repeated  sohition  in  water  and  precipitation  with  ammonium  sulphate  a 
part  of  the  globulin  becomes  insoluble  (Langstein)  .  This  also  occurs  on 
precipitation  by  diluting  with  water  or  by  dialysis,  and  it  is  cpite  possible 
that  the  globulin  is  a  mixture.  That  portion  which  readily  becomes 
insoluble  seems  to  be  identical  with  Eichholz's  glucoproteid  or  Osborne 
and  Campbell's  ovomucin.  Langstein  obtained  11  per  cent  of  glucos- 
amine from  the  soluble  ovoglobulin.  The  total  quantity  of  globulins, 
according  to  Dillner,  is  about  6.7  per  cent  of  the  total  protein  substances, 
and  this  corresponds  with  the  recent  determinations  of  Osborne  and 
Campbell.  In  regard  to  the  probable  occurrence  of  several  globulins 
in  the  white  of  the  egg  there  are  the  statements  of  Corin  and  Berard  as 
well  as  of  Langstein,^  but  they  have  not  led  to  any  positive  conclusions. 

'  Cited  from  v.  Gorup-Besanez,  Lehrbuch,  4.  Aufl.,  739. 

'  Pfluger's  Arch.,  31,  33,  and  39. 

'Langstein,  Hofmeister's  Beitrage,  1;  Eichholz,  Journ.  of  Physiol.,  23;  Osborne 
and  Campbell,  Connecticut  Agric.  Kxp.  Station,  23d  Ann.  Report,  New  Haven,  1900; 
Dillner,  Maly's  Jahresber.,  15;  Corin  and  Berard,  ibid.,  IS. 


OVALBUMIN.  507 

Ovalbumin.  The  so-called  albumin  of  the  egg-white  is  undoubtedh'^ 
a  mixture  of  at  least  two  albumin-like  glucoprotcid-^.  The  views  differ 
considerably  in  regard  to  the  number  of  these  compound  proteids  (Bond- 
ZYNSKi  and  Zoja,  Gautier,  Bechamp,  Corin  and  Berard,  Panormoff, 
and  others).  Since  Hofmeister  has  been  able  to  prepare  ovalbumin  in  a 
crystaUine  form,  and  since  Hopkins  and  Pinkus  ^  have  shown  that  not 
more  than  one-half  of  the  ovalbumin  can  be  obtained  in  such  a  form, 
OsBORXE  and  Campbell  have  isolated  two  different  ovalbumins  or  chief 
fractions;  the  crystallizable  the}"  call  ovalbumin  and  the  non-cr}stallizable 
conalhumin.  The  two  fractions  have  only  a  slight  variation  in  elementary 
composition;  the  conalbumin  coagulates  between  50-60°  C,  nearer  to  60° 
C,  and  the  ovalbumin  at  64°  C.  or  at  a  higher  temperature.  There  are  no 
conclusive  investigations  as  to  whether  the  non-crystallizable  conalbumin 
is  a  mixture  or  not,  and  the  question  concerning  the  unity  of  the  crj'stal- 
lizable  ovalbumin  is  also  disputed.  According  to  Boxdzynski  and  Zoja, 
cr^'staUizable  ovalbumin  is  a  mixture  of  several  albumins  ha\'ing  somewhat 
different  coagulation  temperatures,  solubiliti:^?,  and  specific  rotations, 
while  Hofmeister  and  Laxgsteix  on  the  contrary  believe  that  cry^- 
tallizable  ovalbumin  is  a  unit.  The  statements  as  to  the  specific  rotation 
of  the  different  fractions  unfortunately  differ,  and  the  elementary  analyses 
have  also  given  no  positive  results,  as  a  variation  of  1.2-1.7  per  cent  has 
been  observed  in  the  quantity  of  sulphur.  According  to  the  consistent 
analyses  of  Osborxe  and  Campbell  and  of  Laxgsteix,  the  conalbumin  con- 
tains about  1.7  per  cent  sulphur  and  about  16  per  cent  nitrogen,  while  the 
ovalbumin  contains  on  an  average  about  15.3  per  cent  nitrogen.  Laxg- 
steix ^  obtained  10-11  per  cent  glucosamine  from  ovalbumin  and  about 
9  per  cent  from  conalbumin.  The  ovalbumin,  like  the  conalbumin,  has 
the  properties  of  the  albumins  in  general,  but  differs  from  seralbumin  in 
the  following:  The  specific  rotation  is  lower.  It  is  made  quickly  insoluble 
by  alcohol  and  is  precipitated  by  a  sufficient  quantity  of  HCl,  but  dissolves 
in  an  excess  of  acid  with  greater  difficulty  than  the  seralbumin.  The 
products  isolated  by  Abderhalden  and  Pregl^  on  the  hydrolysis  of 
ovalbumin  do  not  show  anyihing  of  special  interest. 

In  preparing  crj'stalline  ovalbumin  mix,  according  to  Hofmeister,  the 
beaten  white  of  egg  free  from  foam  with  an  equal  volume  of  a  saturated 
ammonium-sulphate  solution,  filter  off  the  globulin,  and  allow  the  filtrate 
to  slowly  evaporate  in  thin  layers  at  the  temperature  of  the  room.  After 
a  time  the  masses  which  separate  out  are  dissolved  in  water,  treated  with 

*  Hofmeister,  Zeitschr.  f.  physiol.  Chem.,  14,  16,  and  24;  Gabriel,  ihid.,  15;  Bond- 
zynski  and  Zoja,  ibid.,  19;  Gautier,  Bull.  Soc.  chim.,14;  Bechamp,  ibid.,  21;  Corin 
and  Berard,  1.  c;  Hopkins  and  Pinkus,  Ber.  d.  d.  chem.  Gesellsch.,  31,  and  Journ.  of 
Physiol.,  23;  Osborne  and  Campbell,  1.  c.;-  Panormoff,  Maly  s  Jahresber.,  27  and  28. 

^Zeitschr.  f.  physiol.  Chem.,  31. 

'/?)Z(i.,46. 


508  ORGANS  OF  GENERATION. 

ammonium-sulphate  solution  until  they  begin  to  get  cloudy,  and  allowed 
to  stand.  After  repeated  recrystallization  the  mass  is  either  treated  with 
alcohol,  which  makes  the  crystals  insoluble,  or  they  are  dissolved  in  water 
and  purified  by  dialysis.  From  these  solutions  the  proteid  does  not  crys- 
tallize again  on  spontaneous  evaporation.  (See  also  page  507,  foot-note  1, 
for  the  Hopkins  and  Pixkus  method.) 

Conalbumin  can  be  removed  from  the  filtrate,  after  the  complete  crys- 
tallization of  the  ovalbumin,  by  removing  the  sulphate  by  means  of  dialysis 
and  coagulating  by  heat. 

Gautier  '  found  a  fibrinogen-like  substance  in  the  white  of  the  egg,  which 
was  changed  into  a  fibrin-like  body  by  the  action  of  a  ferment. 

Ovomucoid.  This  substance,  first  observed  by  Neumeister  and  consid- 
ered by  him  as  a  pseudopeptone  and  then  later  studied  by  Salkowski,  is, 
according  to  C.  Th.  Morner,^  a  mucoid  with  12.65  per  cent  nitrogen  and 
2.20  per  cent  sulphur.  On  boiling  with  dilute  mineral  acids  it  yields  a 
reducing  substance.  Ovomucoid  exists  in  hens'  eggs  to  the  extent  of  about 
10  per  cent  of  the  total  solids. 

A  solution  of  ovomucoid  is  not  precipitated  by  mineral  acids  nor  by 
organic  acids,  with  the  exception  of  phosphotungstic  acid  and  tannic  acid. 
It  is  not  precipitated  by  metallic  salts,  but  basic  lead  acetate  and  ammonia 
render  it  insoluble.  Ovomucoid  is  thrown  do\\Ti  by  alcohol,  but  sodium 
chloride,  sodium  sulphate,  and  magnesium  sulphate  give  no  precipitates 
either  at  the  ordinaiy  temperature  or  when  the  salts  are  added  to  saturation 
at  30°  C.  Its  solutions  are  not  precipitated  by  an  equal  volume  of  a  satu- 
rated solution  of  ammonium  sulphate,  but  are  precipitated  on  adding  more 
salt  thereto.  The  substance  is  not  precipitated  on  boiling,  but  the  part 
which  has  become  insoluble  in  cold  water  which  has  been  dried  is  dissolved 
by  boiling  water.  Zaxetti  has  prepared  glucosamine  on  splitting  ovomucoid 
with  concentrated  hydrochloric  acid,  and  Seemann  found  that  the  quantity 
of  glucosamine  in  ovomucoid  was  34.9  per  cent.^ 

Ovomucoid  may  be  prepared  by  removing  all  the  proteins  by  boiling 
with  the  addition  of  acetic  acid  and  then  concentrating  the  filtrato  and 
precipitating  with  alcohol.  The  substance  is  purified  by  repeated  solution 
in  water  and  precipitation  with  alcohol. 

According  to  Panormow  '  the  eggs  of  other  birds,  such  as  the  pigeon  and 
ducks,  contain  a  special  protein  in  the  egg-white,  which  is  not  identical  with 
that  of  the  hen's  egg. 


'  Compt.  rend.,  13.^. 

2  R.  Neumeister,  Zeitschr.  f.  Biologic,  27;  Salkowski,  Centralbl.  f.  d.  med.  Wis- 
sensch.,  1893,  513  and  706;  C.  Morner,  Zeitschr.  f.  physiol.  Chem.,  18.  See  also  Lang- 
stein,  Hofmeister's  Beitriige,  3  (literature). 

3Zanetti,  Chem.  Centralbl.,  1898,  1;  Seemann,  cited  from  Langstein,  Ergebnisse 
derPhy.siol.,1,  Abt.  1,86. 

*  See  Biochem.  Centralbl.  5 


SHELL  MEMBRANE  AND  EGG   SHELL.  509 

The  mineral  bodies  of  the  white  of  the  egg  have  been  analyzed  by  Poleck 
and  Weber.i  They  found  in  1000  parts  of  the  ash:  276.6-284.5  grams 
potash,  235.6-329.3  soda,  17.4-29  Ume.  17-31.7  magnesia,  4.4-5.5  iron 
oxide,  238.4-285.6  chlorine,  31.6-4S.3  phosphoric  acid  (P2O5),  13.2-26.3 
sulphmic  acid,  2.8-20.4  silicic  acid,  and  96.7-116  grams  carbon  dioxide. 
Traces  of  fluorine  have  also  been  found  (Nickles^).  The  ash  of  the  white 
of  the  egg  contahis,  as  compared  •v\ith  the  yolk,  a  greater  amount  of  chlorine 
and  alkalies  and  a  smaller  amount  of  lime,  phosphoric  acid,  and  iron. 

The  Shell-membrane  and  the  Egg-shell.  The  shell-membrane  consists, 
as  above  stated  (page  73),  of  a  keratin  substance.  The  shell  contains  very 
little  organic  substance,  36-65  p.  m.  The  chief  mass,  more  than  900  p.  m., 
consists  of  calcium  carbonate;  besides  this  there  are  verj^  small  amounts  of 
magnesium  carbonate  and  earthy  phosphates. 

The  diverse  coloring  of  birds'  eggs  is  due  to  several  different  coloring -matters. 
Among  these  we  find  a  red  or  reddish-brown  pigment  called  "  oorodein  "  by  Sorby,^ 
which  is  perhaps  identical  with  haematoporphyrin.  The  green  or  blue  coloring- 
matter,  Sorby's  oocyan,  seems,  according  to  Liebermanx  *  and  Krukenberg,^ 
to  be  partly  hiliverdin  and  partly  a  blue  derivative  of  the  bile-pigments. 

The  eggs  of  birds  have  a  space  at  their  blunt  end  filled  \\ith  gas;  this 
gas  contains  on  an  average  18.0-19.9  per  cent  oxygen  (Hufxer  ^). 

The  weight  of  a  hen's  egg  varies  between  40-60  grams  and  may  some- 
times reach  70  grams.  The  shell  and  shell-membrane  together,  when  care- 
fully cleaned,  but  still  in  the  moist  state,  weigh  5-8  grams.  The  yolk 
weighs  12-18  and  the  white  23-34  grams,  or  about  double.  The  entire  egg 
contains  2.8-7.5,  or  average  4.6,  milligrams  of  iron  oxide,  and  the  quantity 
of  iron  can  be  increased  by  food  rich  in  iron  (Hartung  ''). 

The  white  of  the  egg  of  cartilaginous  and  bony  fishes  contains  only  traces  of 
true  albumin,  and  the  cover  of  the  frog's  egg  consists,  according  to  Giacosa,  of 
mucin.  The  eggs  of  the  river-perch  contain,  according  to  Hammarsten,^  mucin 
in  the  envelope  in  the  unripe  state  and  only  mucinogen  in  the  ripe  state.  The 
crystalline  formations  (ijolk-sphcrules,  or  dotterpldttchen)  which  have  been  observed 
in  the  egg  of  the  tortoise,  frog,  ray,  shark,  and  other  fishes,  and  which  are  de- 
scribed by  Valexciexxes  and  Fremy^  under  the  names  emydin,  ichthin,  ichthidin, 
and  ichthidin,  seem,  as  above  stated  in  connection  with  ichthulin,  to  consist  chiefly 
of  phosphoglucoproteids.  The  eggs  of  the  river-crab  and  the  lobster  contain  the 
same  pigment  as  the  shell  of  the  animal.  This  pigment,  called  cyanocrystallinf 
becomes  red  on  boiling  in  water. 

'  Cited  from  Hoppe-Seyler,  Physiol.  Chem..  778. 

^  Compt.  rend.,  43. 

^  Cited  from  Krukenberg,  Verh.  d.  phys.-chem.  Gesellsch.  in  Wiirzburg,  17. 

*  Ber.  d.  deutsch.  chem.  Gesellsch.,  11. 
»1.  c. 

•Arch.  f.  (Anat.  u.)  Physiol.,  1892. 
1  Zeitschr.  f .  Biologie,  43. 

*  Giacosa,  Zeitschr.  f.  physiol.  Chem.,  7,-  Hammarsten,  Skand.  An-h.  f.  Physiol.,  17. 
'Cited  from  Hoppe-Seyler' s  Physiol.  Chem.,  77. 


510  ORGANS  OF  GENERATION. 

C.  i\IoRNER  *  has  isolated  a  substance  which  he  calls  j^crcaylobulin  from  the 
unripe  eggs  of  the  river-perch.  It  is  a  globulin  and  has  a  strong  astringent  taste. 
Especially  striking  is  its  property  of  precipitating  certain  glucoproteids,  such  as 
ovomucoid  and  ovarial  mucoids,  and  polysaccharides,  such  as  glycogen,  gum 
tragacanth  or  quince-seed  gum,  and  starch-paste,  and  of  being  precipitated  by 
them. 

In  fossil  eggs  (of  aptenodytes,  pelecanus,  and  hali^us)  in  old  guano 
deposits,  a  yellowdsh  white,  silky,  laminated  compound  has  been  found  which 
is  called  guanovulit,  (NHJlSOi +2K2SO4 +3KHSO4 +4H2O,  and  which  is  easily 
soluble  in  water,  but  is  insoluble  in  alcohol  and  ether. 

Those  eggs  which  develop  outside  of  the  mother-organism  must  con- 
tain all  the  elements  necessary  for  the  yomig  animals.  One  finds,  therefore, 
in  the  yolk  and  white  of  the  egg  an  abundant  quantity  of  protein  bodies  of 
different  kinds,  and  especially  phosphorized  proteins  in  the  yolk.  Further, 
we  also  find  lecithin  in  the  yolk,  which  seems 'habitually  to  occur  in  the 
developing  cell.  The  occurrence  of  glycogen  is  doubtful,  and  the  carbo- 
hydrates are  perhaps  represented  by  a  very  small  amomit  of  sugar  and 
glucoproteids.  On  the  contrary,  the  egg  contains  a  large  proportion  of  fat, 
which  doubtless  is  important  as  a  source  of  supply  of  nourishment  and 
in  maintaining  respiration  for  the  embtyo.  The  cholesterin  and  the  lutein 
can  hardly  have  a  direct  influence  on  the  development  of  the  embr}^o. 
The  egg  also  seems  to  contain  the  mineral  bodies  necessar}^  for  the  develop- 
ment of  the  young  animal.  The  lack  of  phosphoric  acid  is  com]5ensated 
by  an  abundant  amount  of  phosphorized  organic  substance,  and  the 
nucleoalbumin  containing  iron,  from  which  the  hsematogen  (see  page  5C3) 
is  formed,  is  doul^tless,  as  Bunge  claims,  of  great  importance  in  the  forma- 
tion of  the  haemoglobin  containing  iron.  The  silicic  acid  necessary  for  the 
development  of  the  feathers  is  also  found  in  the  egg. 

During  the  period  of  incubation  the  egg  loses  weight,  chiefly  due  to 
loss  of  water.  The  quantity  of  solids,  especially  the  fat  and  the  proteins, 
diminishes,  and  the  egg  gives  off  not  onl}^  carbon  dioxide,  but  also,  as 
LiEBERMANN  ^  has  shown,  nitrogen  or  a  nitrogenous  substance.  The  loss 
is  compensated  by  the  absorption  of  oxygen,  and  it  is  found  that  during 
incubation  a  respiratory  exchange  of  gases  takes  place. 

As  Bohr  and  Hasselbai.ch  have  shown  by  exact  investigations,  the 
elimination  of  carbon  dioxide  is  very  small  in  the  first  days  of  incubation; 
on  the  fourth  day  the  carbon-dioxide  production  gradually  increases,  and 
after  the  ninth  day  it  augments  in  the  same  proportion  as  the  weight  of 
the  foetus.  Calculated  upon  1  kilogram  weight  for  one  hour  it  is,  from  the 
ninth  day  on,  about  tlie  same  as  in  the  full-groAvn  hen.     Hasselbalch^ 


'  Zeitschr.  f.  physiol.  Chem.,  40. 
*  Pfliiger's  Arch.,  43. 

'Bohr    and    Plasselbalch,    Maly's  Jahresber.,    29;    Hasselbaleh,  Skand.    Arch.  f. 
Physiol.,  13. 


INCUBATION   OF  THE   EGG.  511 

has  also  sho^^-n  that  the  fertilized  hen's  egg  not  only  gives  off  nitrogen  the 
tirst  five  or  six  hours  of  incubation,  but  also  some  oxygen,  and  that  we 
are  here  dealing  with  an  oxygen  production  which  runs  parallel  with  the 
cell-di\asion.  It  is  not  kno'UTi  whether  this  oxygen  formation  connected 
with  the  life  of  the  cell  is  a  fermentative  or  a  so-called  vital  process. 

\Miile  the  quantity  of  drj'  substance  in  the  egg  during  this  period  always 
decreases,  the  quantity  of  mineral  bodies,  protein,  and  fat  always  increases 
in  the  embr^^o.  The  increase  in  the  amount  of  fat  in  the  embrj'O  depends, 
according  to  Liebermann,  in  great  part  upon  a  taking  up  of  the  nutritive 
yolk  in  the  abdominal  cavity.  The  weight  of  the  shell  and  the  quantity  of 
lime-salts  contained  therein  remain  michanged  during  incubation.  The 
yolk  and  white  together  contain  the  necessary  quantity  of  lime  for  devel- 
opment. 

The  most  complete  and  careful  chemical  mvestigation  on  the  develop- 
ment of  the  embrv^o  of  the  hen  has  been  made  by  Liebermaxn.  From  his 
researches  we  may  quote  the  folloAiNing:  In  the  earlier  stages  of  the  devebp- 
ment,  tissues  verj-  rich  in  water  are  formed,  but  upon  the  continuation  of 
the  development  the  quantity  of  water  decreases.  The  absolute  quantity 
of  the  bodies  soluble  in  water  increases  with  the  devek)pment,  while  their 
relative  quantity,  as  compared  "^ith  the  other  solids,  continually  decreases. 
The  quantity  of  the  bodies  soluble  in  alcohol  quickly  increases.  A  specially 
important  increase  is  noticed  in  the  fat,  whose  quantity  is  not  very  great 
even  on  the  fourteenth  day,  but  after  that  it  becomes  considerable.  The 
quantity  of  protein  bodies  and  albuminoids  soKible  in  water  grows  contin- 
ually and  regularly  in  such  a  way  that  their  absolute  quantity  increases, 
while  their  relative  quantity  remains  nearly  unchanged.  Liebermann 
found  no  gelatine  in  the  embrj'O  of  the  hen.  The  embrj^o  does  not  contain 
any  gelatine-forming  substance  until  the  tenth  day,  and  from  the  fourteenth 
day  on  it  contains  a  body  which,  when  boiled  with  water,  gives  a  substance 
similar  to  chondrin.  A  body  similar  to  mucin  occurs  in  the  embrj'o  when 
about  six  days  old,  but  then  disappears.  The  quantity  of  haemoglobin 
shows  a  continual  increase  compared  with  the  weight  of  the  body.  Lieber- 
mann found  that  the  relationship  of  the  haemoglobin  to  the  body  weight 
was  1 :728  on  the  eleventh  day  and  1 :421  on  the  twenty-first  day. 

By  means  of  Berthelot's  thermometric  methods  Tangl  has  deter- 
mined the  chemical  energy  present  at  the  beginning  and  end  of  the  develop- 
ment of  the  embrj'o  of  the  sparrow's  and  hen's  eggs.  The  difference  was 
considered  as  work  of  development.  He  found  that  the  chemical  energy 
necessary  for  the  development  of  1  gram  of  ripe  or  nearly  ripe  hen's  embr\o 
(Plymouth  egg)  was  equal  to  658  calories.  Tliis  energ}'  originated  chiefly 
from  the  fat.  Of  the  total  chemical  energ}-  utilized,  two-thirds  was  used 
for  the  construction  of  the  embryo-  and  one-third  transformed  into  other 
forms  of  energy  as  work  of  development.     Still  more  recent  researches  of 


512  ORGANS  OF  GENERATION. 

Bohr  and  Hasselbalch  ^  show  that  none  of  the  transformed  chemical 
energy  is  used  in  the  construction  of  the  embr}'o,  as  it  leaves  the  egg  almost 
entirely  as  heat. 

By  their  investigations  on  the  development  of  the  trout  egg  Tangl  and 
Farkas-  have  found  that  the  loss  in  weight  of  each  egg  which  had  an  aver- 
age weight  of  88  milligrams  was  4.9  milligrams  during  the  42  days  of 
incubation,  of  which  4.11  milligrams  was  water  and  0.722  milligram  dry 
substance  with  0.367  milligram  C.  The  eggs  loose  no  nitrogen  and  no  fat. 
The  fat  content  increases  a  little,  and  indeed,  as  these  authors  believe, 
at  the  expense  of  the  proteins.  The  chemical  energy  used  during  develop- 
ment was  6.68  gram-calories. 

The  tissue  of  the  placenta  has  not  thus  far  been  the  subject  of  detailed  chemical 
investigation.  It  contains  a  protein  which  coagulates  at  60-65°  (Bottazzi  and 
Delfino),  also  glycocoll  and  a  proteolytic  as  well  as  a  diastatic  enzyme  (Ascoli, 
Raineri,  Bergell,  and  Liepmann  0-  In  the  edges  of  the  placenta  of  bitches 
and  of  cats  a  crystallizable  orange-colored  pigment  (bilirubin?)  has  been  found, 
and  also  a  green  amorphous  pigment,  whose  relationship  to  biliverdin  is  not 
kno^ATi.'' 

From  the  cotyledons  of  the  placenta  in  rimiinants  a  white  or  faintly  rose-colored 
creamy  fluid,  the  uterine  milk,  can  be  obtained  by  pressure.  It  is  alkaline  in 
reaction,  but  becomes  acid  quickly.  Its  specific  gra\dty  is  1.033-1.040.  It  con- 
tains as  form-elements  fat-globules,  small  granules,  and  epithelium-cells.  There 
have  been  found  81.2-120.9  p.m.  solids,  61.2-105.6  p.m.  protein,  about  10  p.  m. 
fat,  and  3.7-8.2  p.  m.  ash  in  the  uterine  milk. 

The  fluid  occurring  in  the  so-called  grape-mole  (Mola  racemosa)  has  a  low 
specific  gravity,  1.009-1.012,  and  contains  19.4-26.3  p.  m.  solids  ^\ath  9-10  p.m. 
protein  bodies  and  6-7  p.  m.  ash. 

The  amniotic  fluid  in  women  is  thin,  whitish,  or  pale  yellow;  sometimes 
it  is  somewhat  yellowish  brown  and  cloudy.  White  flakes  separate.  The 
form-elements  are  mucus-corpuscles,  epithelium-cells,  fat-drops,  and  lanugo 
hair.  The  odor  is  stale,  the  reaction  neutral  or  faintly  alkaline.  The 
specific  gravity  is  1.002-1.028. 

The  amniotic  fluid  contains  the  constituents  of  ordinar}^  transudates. 
The  amount  of  solids  at  birth  is  hardly  20  p.  m.  In  the  earlier  stages  of 
pregnancy  the  fluid  contains  more  solids,  especially  proteins.  Among  the 
protein  bodies  Weyl  found  one  substance  similar  to  vitellin,  and  with  great 
probability  also  seralbumin,  besides  small  quantities  of  mucin.  Enzymes 
of  various  kinds  (pepsin,  diastase,  thrombin,  lipase)  occur,  according  to 
BoNDi.  Sugar  is  regularly  found  in  the  amniotic  fluid  of  cows,  but  not  in 
human  beings.  In  the  ox,  pig,  and  goat  Gurber  and  GrIixbaum  have  also 
found  levulose.     The  human  amniotic  fluid  also  contains  some  urea,  uric 

'Tangl,  Pfliiger's  Arch.,  93;    Bohr  and  Hasselbalch,  Skand.  Arch.  f.  Physiol.,  14. 
2  Pfluger's  Arch.,  104. 

^Bottazzi  and  Delfino,  Centralbl.  f.  Physiol.,  18,  114;    Ascoli,  ibid.,  Ifi;    Raineri, 
Biochem.  Centralbl.,  4,  428;   Bergell  and  Liepmann,  Munch,  med.  Wochenschr.,  1905. 
*See  Etti,  Maly's  Jahresber.,  2,  287,  and  Preyer,  Die  Blutkristalle,  Jena,  1871. 


AMNIOTIC    FLUID.  513 

a/yid,  and  allantoin.  The  quantity  of  these  may  be  mcreased  in  hydramnion 
(Prochowxick,  IIarxack),  which  depends  on  an  increased  secretion  by 
the  kidneys  and  skin  of  the  foetus.  Creatine  and  lactates  are  doubtful 
constituents  of  the  amniotic  fluid.  The  quantity  of  urea  in  the  amniotic 
fluid  is,  according  to  Prochowxick,  0.16  p.  m.  In  the  fluid  in  hydramnion 
Prochowxick  and  Harxack  found  respectively  0.34  and  0.48  p.  m.  urea. 
The  chief  mass  of  the  solids  consists  of  salts.  The  .quantity  of  chlorides 
(NaCl)  is  5.7-6.6  p.  m.  The  molecular  concentration  of  the  amniotic  fluid 
is  somewhat  lower  than  that  of  the  blood,  which  is  no  doubt  due  to  a  dilu- 
tion by  the  foetal  urine  (Zaxgemeister  and  Meissl  i). 

»Weyl,  Arch.  f.  (.\nat.  u.)  Physiol.,  1S76;  Bondi,  Centralbl.  f.  Gynakol.,  1903; 
Prochownick.  Arch.  f.  Gyniik.,  11,  also  Maly's  Jahresber.,  7,  155;  Harnack,  Berlin, 
klin.  Wochenschr.,  1888  No.  41;  Zangemeister  and  Meissl,  Munch,  med.  Wochenschr., 
'1903;  Giirber  and  Grunbaum,  Und.,  1904. 


CHAPTER  XIV. 

MILK. 

The  chemical  constituents  of  the  mammary  glands  have  been  little 
studied.  The  cells  are  rich  in  protein  and  nucleo-proteids.  Among  the  latter 
we  have  one  that  yields  pentose  and  guanine,  but  no  other  purine  base,  on 
boiling  with  dilute  mineral  acids.  This  com'pound  proteid,  investigated  by 
Odenius,  contains  as  an  average  the  following:  17.28  per  cent  N,  0.89  per 
cent  S,  and  0.277  per  cent  P.  Besides  this  proteid  we  have  at  least  one 
other,  as  Maxdel  and  Levexe  and  Loebisch^  have  isolated  a  nucleic  acid 
from  the  mammary  gland,  which,  like  the  thymonucleic  acids,  yielded  ade- 
nine, guanine,  thymine,  and  cytosine.  This  nucleic  acid  also  gave  the  pen- 
tose reactions  and  yielded  abundance  of  levulinic  acid.  Besides  this  nucleic 
acid.  Maxdel  and  Levexe  ~  isolated  from  the  glands  aglucothionic  acid  ^^ith 
2.65  per  cent  S  and  4.38  per  cent  N.  We  cannot  state  what  relation  these 
substances  bear  to  that  constituent  of  the  gland  foimd  by  Bert,  which  on 
boiling  with  dilute  mineral  acids  yielded  a  reducing  substance.  A  similar 
substance,  wiiich  acts  perhaps  as  a  step  towards  the  formation  of  lactose, 
has  also  been  observ^ed  by  Thierfelder.  It  is  to  be  expected  that  these 
bodies  are  steps  in  the  formation  of  milk-sugar;  still  we  have  no  point  of 
support  for  such  an  assumption,  and  the  recent  investigations  seem  to 
indicate  that  the  milk-sugar  is  produced  in  the  glands  by  a  transformation 
of  the  sugar  of  the  blood.  Fat  seems,  at  least  in  the  secreting  glands,  to 
be  a  never-failing  constituent  of  the  cells,  and  this  fat  may  be  observed  in 
the  protoplasm  as  large  or  small  globules  similar  to  milk-globules.  The 
extractive  bodies  of  the  mammary  glands  have  been  little  investigated,  but 
among  them  a:e  found  considerable  amounts  of  purine  bases.  The  mam- 
mary glands  contain  also  a  proteolytic  enzyme  which,  according  to  Hilde- 
nuAXDT,3  occurs  to  a  much  greater  extent  in  the  active  gland  as  compared 
with  the  inactive  one. 


'  Odenius,  Maly'.s  Jahre.sber.,  30;    Mandel  and  Levene,  Zeitschr.  f.  physiol.  Chem.^ 
46;  Loebisch,  HofmeLster's  Beitrage,  8. 

^  Zeitschr.  f.  physiol.  Chem.,  4.5. 

^  Bert,  Compt.  rend.,  98;   Thierfelder,  Pfliiger's  Arch.,  34,  and  Maly's  Jahresber. 
13;    Hildebrandt,  Hofmeister's  Beitrage,  5. 

514 


COW'S   MILK.  515 

As  human  milk  and  the  milk  of  animals  are  essentially  of  the  same 
constitution,  it  seems  best  to  speak  first  of  the  one  most  thoroughly  inves- 
tigated, namely,  cow's  milk,  and  then  of  the  essential  properties  of  the 
remaining  important  kinds  of  milk.^ 

Cow's  Milk. 

Cow's  milk,  like  every  other  kind,  forms  an  emulsion  which  consists  of 
very  finely  divided  fat  suspended  in  a  solution  consisting  chiefly  of  protein 
bodies,  milk-sugar,  and  salts.  Milk  is  non-transparent,  white,  whitish 
yellow,  or  in  thin  layers  somewhat  bluish  white,  of  a  faint,  insipid  odor  and 
mild,  faintly  sweetish  taste.  The  specific  gravity  is  1.028  to  1.0345  at 
15°  C.  The  freezing-point  is  0.54-0.59°  C,  average  0.563°  C,  and  the 
molecular  concentration  0.298. 

The  reaction  of  perfectly  fresh  milk  is  generally  amphoteric  towards 
litmus.  The  extent  of  the  acid  and  alkaline  part  of  this  amphoteric  reac- 
tion has  been  determined  by  different  investigators,  especially  Thorxer, 
Sebelien,  and  Courant.^  The  results  differ  somewhat  with  the  indicators 
used,  and  moreover  the  milk  from  different  animals,  as  well  as  that  from 
the  same  animal  at  different  times  during  the  lactation  period,  varies 
somewhat.  Courant  has  determined  the  alkaline  part  by  N/10  sulphuric 
acid,  using  blue  lacmoid  as  indicator,  and  the  acid  part  by  N/10  caustic 
soda,  using  phenolphthalein  as  indicator.  He  found,  as  an  average  for  the 
first  and  last  portions  of  the  milking  of  twenty  cows,  that  100  c.c.  milk 
had  the  same  alkaline  reaction  toward  blue  lacmoid  as  41  c.c.  N/10 
caustic  soda,  and  the  same  acid  reaction  toward  phenolphthalein  as 
19.5  c.c.  N/10  sulphuric  acid.  The  actual  reaction  of  cow's  milk,  which 
follows  from  the  electrometric  estimation,  is,  on  the  contrary,  according  to 
FoA,-*^  nearly  neutral,  like  the  reaction  of  animal  fluids  and  tissues  in  general. 

Milk  gradually  changes  when  exposed  to  the  air,  and  its  reaction 
becomes  more  and  more  acid.  This  depends  on  a  gradual  transformation 
of  the  milk-sugar  into  lactic  acid,  caused  by  micro-organisms. 

Perfectly  fresh  amphoteric  milk  does  not  coagulate  on  boiling,  but  forms 
a  pellicle  consisting  of  coagulated  casein  and  lime-salts,  which  rapidly  re- 
forms after  being  removed.  Even  after  passing  a  current  of  carbon  dioxide 
through  the  fresh  milk  it  does  not  coagulate  on  boiling.  In  proportion 
as  the  formation  of  lactic  acid  advances  this  behavior  changes,  and  soon  a 

*  A  very  complete  reference  to  the  literature  on  milk  may  be  found  in  Raudnitz's 
"Die  Bestandteile  der  Milch,"  in  Ergebnisse  der  Physiol.,  2,  Abt.  1.  The  literature 
of  the  last  few  years  may  be  found  in  the  references  by  Raudnitz,  Monatsschrift  f. 
Kinderheilkunde. 

^  Thorner,  Maly's  Jahresber.,  22;   Sebelien,  ibuL;   Courant,  Pfliiger's  Arch.,  50. 

'Compt.  rend.  Soc.  biolog.  (58),  59,  51. 


516  MILK. 

stage  is  reached  when  the  milk,  which  has  previously  had  carbon  dioxide 
passed  through  it,  coagulates  on  boiling.  At  a  second  stage  it  coagulates 
alone  on  heating;  then  it  coagulates  by  passing  carbon  dioxide  alone  with- 
out boiling;  and  lastly,  when  the  formation  of  lactic  acid  is  sufficient,  it 
coagulates  spontaneously  at  the  ordinary  temperature,  forming  a  solid 
mass.  It  may  also  happen,  especially  in  the  warmth,  that  the  casein- 
clot  contracts  and  a  yellowish  or  yellowish-green  acid  liquid  (acid  whey) 
separates. 

Milk  may  undergo  various  fermentations.  Lactic-acid  fermentation,  brought 
about  by  Huppe's  lactic-acid  bacillus  and  also  other  varieties,  takes  first  place. 
Li  the  spontaneous  souring  of  milk  we  generally  consider  the  formation  of  lactic 
acid  as  the  most  essential  product,  but  a  formation  of  succinic  acid  may  also  take 
place,  and  in  certain  bacterial  decompositions  of  milk,  succinic  acid  and  no  lactic 
acid  is  formed.  The  materials  from  which  these  two  acids  are  formed  are  lactose 
and  lactophosphocarnic  acid.  Besides  the  lactic  acids,  the  optically  inactive 
as  well  as  the  dextro  and  levo  acids,  and  succinic  acid,  volatile  fatty  acids,  such 
as  acetic  acid,  butyric  acid,  and  others,  may  be  formed  in  the  bacterial  decompo- 
sition of  milk. 

Milk  sometimes  undergoes  a  peculiar  kind  of  coagulation,  being  converted 
into  a  thick,  ropy,  slimy  mass  (thick  milk).  This  conversion  depends  upon  a 
peculiar  change  in  which  the  milk-sugar  is  made  to  undergo  a  slimy  transforma- 
tion.    This  transformation  is  caused  by  special  micro-organisms. 

If  the  milk  is  sterilized  by  heating  and  contact  with  micro-organisms 
prevented,  the  formation  of  lactic  acid  may  be  entirely  stopped.  The 
production  of  acid  may  also  be  prevented,  at  least  for  some  time,  by  many 
antiseptics,  such  as  saUcyUc  acid,  thymol,  boric  acid,  and  other  bodies. 

If  freshly  drawn  amphoteric  milk  is  treated  with  rennet,  it  coagulates 
quickly,  especially  at  the  temperature  of  the  body,  to  a  solid  mass  (curd) 
from  which  a  yellowish  fluid  (sweet  whey)  is  gradually  pressed  out.  This 
coagulation  occurs  without  any  change  in  the  reaction  of  the  milk,  and 
therefore  it  is  distinct  from  the  acid  coagulation. 

In  cow's  milk  we  find  as  form-elements  a  few  colostrum  corpuscles 
(see  Colostrum)  and  a  few  pale  nucleated  cells.  The  number  of  these 
form-elements  is  very  small  compared  with  the  immense  amount  of  the 
most  essential  form-constituents,  the  milk-globules. 

The  Milk-globules.  These  consist  of  extremely  small  drops  of  fat 
whose  number  is,  according  to  Woll,i  L06-5.75  millions  in  1  c.mm.,  and 
whose  diameter  is  0.0024-0.0046  mm.  and  0.0037  mm.  as  an  average  for 
different  kinds  of  animals.  It  is  unquestionable  that  the  milk-globules 
contain  fat,  and  we  consider  it  as  positive  that  all  the  milk-fat  exists  in 
them.  Another  disputed  question  is  whether  the  milk-globules  consist 
entirely  of  fat  or  whether  they  also  contain  protein. 

*  On  the  Conditions  Influencing  the  Number  and  Size  of  Fat-globules  in  Cow's  Milk, 
"Wisconsin  Exp.  Station,  C,  1892. 


MILK   GLOBULES.  517 

According  to  the  obsen^ations  of  Ascherson/  drops  of  fat,  wiien 
dropped  in  an  alkaline  protein  solution,  are  covered  \\ith  a  fine  albuminous 
coat,  a  so-called  haptogen-membrane.  As  milk  on  shaking  ^^'ith  ether  does 
not  give  up  its  fat,  or  only  very  slowly  in  the  presence  of  a  great  excess 
of  ether,  and  as  this  takes  place  ver>'  readily  after  the  addition  of  acids  or 
alkalies,  which  dissolve  proteins,  it  was  formerly  thought  that  the  fat- 
globules  of  the  milk  were  enveloped  in  a  protein  coat.  A  true  memljrane 
has  not  been  detected;  and  since,  when  no  means  of  dissolving  the  protein 
is  resorted  to — for  example,  when  the  milk  is  precipitated  by  carbon  dioxide 
after  the  addition  of  ver}'  little  acetic  acid,  or  when  it  is  coagulated  by 
rennet — the  fat  can  be  verj'  easily  extracted  by  ether,  the  theor\'  of  a 
special  albuminous  membrane  for  the  fat-globule  has  been  generally  aban- 
doned. The  obsen,'ations  of  Quincke  ^  on  the  behavior  of  the  fat-globules 
in  an  emulsion  prepared  "uith  gum  have  led,  at  the  present  time,  to  the 
conclusion  that  each  fat-globule  in  the  milk  is  surrounded  by  a  stratum  of 
casein  solution  held  by  molecular  attraction,  and  this  prevents  the  globules 
from  miiting  with  each  other.  Everything  that  changes  the  physical 
condition  of  the  casein  in  the  milk  or  precipitates  it  must  necessarily  help 
the  solution  of  the  fat  in  ether,  and  it  is  in  this  way  that  the  alkalies, 
acids,  and  rennet  act. 

V.  Storch  has  sho-^ii,  in  opposition  to  these  views,  that  the  milk-glol> 
ules  are  surrounded  by  a  membrane  of  a  special  slimy  substance.  This 
substance  is  very  insoluble,  contains  14.2-14.79  per  cent  nitrogen,  and  yields 
a  sugar,  or  at  least  a  reducing  substance,  on  boiling  with  hydrochloric  acid. 
It  is  neither  casein  nor  lactalbumin,  but  seems  to  all  appearances  to  be 
identical  ^ith  the  so-called  "stroma  substance"  detected  by  R.u)ex- 
HAUSEN  and  Daxilewsky.  Storch  was  able  to  show,  by  staining  the 
fat-globules  with,  certain  dyes,  that  this  substance  enveloped  them  like  a 
membrane.  Recently  Voltz  has  given  further  proofs  of  the  ^-iew  that 
the  fat-globules  probably  have  a  membrane,  which  according  to  him  is  a 
ver}'  labile  formation  of  variable  composition.  Droop-Richmoxd  and 
BoNNEMA,^  on  the  other  hand,  present  several  reasons  in  opposition  to 
Storch's  view.  If  Storch's  observation  that  the  purified  fat-globules 
contain  a  special  protein  substance  differing  from  the  dissolved  proteins 
of  the  milk  is  correct,  then  the  assumption  as  to  a  special  bod}'  forming 
a  membrane  or  stroma  of  the  fat-globules  becomes  very  probable. 

Tlie  milk-fat  which  is  obtained  imder  the  name  of  butter  consists 
chiefly  of  olein  and  palmitin.     Besides  these  it  contains,  as  triglycerides, 

'  Arch.  f.  Anat.  u.  Physiol.,  1840. 

^Pfluger's  Arch.,  19. 

'  V.  Storch,  see  Maly's  Jahresber.,  2";  Radenhausen  and  Danilewsky,  Forschungen 
auf  dem  Gebiete  der  Viehhaltung  (Bremen,  1880),  Heft  9;  Voltz,  Pfliiger's  Arch.,  102; 
Droop-Richmond,  see  Chem.  Centralbl.,  1904,  2,  356;   Bonnema,  ibid.,  1243. 


518  MILK. 

myristic  acid,  stearic  acid,  small  amounts  of  lauric  acid,  arachidic  acid,  and 
dioxystearic  acid,  besides  butyric  acid  and  caproic  acid,  traces  of  caprylic 
acid  and  capric  acid.  It  must  not  be  accepted  that  triglycerides  of  volatile 
fatty  acids  occur,  but  rather  mixed  triglycerides  of  volatile  and  non-volatile 
fatty  acids  (Riegel).  Milk-fat  also  contains  a  small  quantity  of  lecithin  and 
cholestcrin  and  a  yellow  coloring-matter.  The  quantity  of  volatile  fatty 
acids  in  butter  is,  according  to  Duclaux,  on  an  average  about  70  p.  m., 
of  which  37-51  p.  m.  is  butyric  acid  and  30-33  p.  m.  is  caproic  acid.  The 
non-volatile  fat  consists  of  jo— /o  olein,  and  the  remainder  is  chiefly  palmitin. 
The  composition  of  butter  is  not  constant,  but  varies  considerably  under 
different  circumstances.^  According  to  Lemus^  the  small  fat-globules 
contain  more  olein  and  less  volatile  acids  than  the  large  globules. 

The  milk-plasma,  or  that  fluid  in  which  the  fat-globules  are  suspended, 
contains  several  different  proteins,  the  statements  as  to  the  number  and 
nature  of  which  are  somewhat  at  variance.  The  three  following,  casein, 
lactalbumin,  and  lactoglohulin,  have  been  closest  studied  and  are  well  char- 
acterized. The  milk-plasma  contains  two  carbohydrates,  of  which  the 
one,  lactose,  is  of  great  importance.  It  also  contains  extractive  bodies, 
traces  of  urea,  creatine,  creatinine,  orotic  acid,  hypoxanthine  (?),  lecithin, 
cholestcrin,  citric  acid  (Soxhlet  and  Henkel^),  and  lastly  also  mineral 
bodies  and  gases. 

Casein.  This  protein  substance,  which  thus  far  has  been  detected  posi- 
tively only  in  milk,  belongs  to  the  nucleoalbumins,  and  differs  from  the 
albuminates  chiefly  by  its  content  of  phosphorus  and  by  its  behavior  with 
the  rennet  enzyme.  Casein  from  cow's  milk  nas  the  following  composition: 
C  53.0,  H  7.0,  N  15.7,  S  0.8,  P  0.85,  and  0  22.65  per  cent.  Its  specific 
rotation  is,  according  to  Hoppe-Seyler,  somewhat  variable;  in  neutral 
solution  it  is  {a)j)=—80°;  its  faintly  alkaline  solution  has  a  stronger  rota- 
tion, namely,  -97.8  to~111.8°,  in  a  solution  of  N/lO-N/5  NaOH  (Long  4). 
The  question  whether  the  casein  from  different  kinds  of  milk  is  identical 
or  whether  there  are  several  different  caseins  is  still  disputed. 

Casein  when  dry  appears  like  a  fine  white  powder,  which  has  no  meas- 
urable solubility  in  pure  water  (Laqueur  and  Sackur).  Casein  is  only 
very  slightly  soluble  in  the  ordinary  neutral-salt  solutions.     According  to 

*  Riegel,  Maly's  Jahresber.,  34;  Duclaux,  Compt.  rend.,  104.  Various  statements 
as  to  the  composition  of  milk-fat  can  be  found  in  Koefoed,  Bull.d.  1'  Acad.  Roy. 
Danoise,  1891,  and  Wanklyn,  Chemical  News,  63;  Browne,  Chem.  Centralbl.,  1899,  2, 
883. 

^  See  Maly's  Jahresber.,  34. 

'■'  Cited  from  F.  Soldner,  Die  Salze  der  Milch,  etc.,  Landwirth.sch.  Ver.suchsstation, 
35,  Separatabzug,  18. 

*  Hoppe-Seyler,  Handl).  d.  physiol.  u.  pathol.  chem.  Analyse,  7.  Aufl.,  368;  Long, 
Journ.  Amer.  Chem.  Soc.  27. 


CASEIN.  519 

Arthus  it  dissolves  rather  easily  in  a  1  per  cent  solution  of  sodium  fluoride, 
ammoniiun  or  potassium  oxalate.  It  is  at  least  a  tetrabasic  acid,  whose 
equivalent  weight  is  1135  according  to  Laqueur  and  Sackur,^  and  whose 
molecular  weight  is  four  or  six  times  this.  The  salts  are  split  hydrolytically. 
It  dissolves  readily  m  water  ^\ith  the  aid  of  alkali  or  alkaline  earths, 
also  calcium  carbonate,  from  which  it  expels  carbon  dioxide.  If  casein 
is  dissolved  in  lime-water  and  this  solution  carefully  treated  with  very 
dilute  phosphoric  acid  until  it  is  neutral  in  reaction,  the  casein  appears  to 
remain  m  solution,  but  is  probably  only  swollen  as  in  milk,  and  the  Kquid 
contains  at  the  same  time  a  large  quantity  of  calcium  phosphate  without 
any  precipitate  or  any  suspended  particles  being  visible.  Tlie  casein  solu- 
tions containing  Ume  are  opalescent  and  have  on  warming  the  appearance 
of  milk  deficient  in  fat  (which  is  also  true  for  the  salts  of  casein  with  the 
alkaline  earths).  Therefore  it  is  not  impossible  that  the  white  color  of  the 
milk  is  due  partly  to  the  casein  and  calcium  phosphate.  Soldxer  has 
prepared  two  calcium  compounds  of  casein  ^ith  1.55  and  2.36  per  cent 
CaO,  and  these  compounds  are  designated  di-  and    tricalcium    casein    by 

COURAXT.  2 

According  to  Laqueur .^  who  has  determined  the  electrical  conduc- 
tivity and  the  internal  friction  of  casein  solutions,  all  casein-salt  solu- 
tions consist  of  a  mixture  of  casein  ions  (with  different  amomits  of  H 
which  can  be  split  off  electrolytically)  and  unsplit  casein  (produced  by 
hydrolysis).  By  the  gradual  addition  of  alkali  to  the  casein  he  found  no 
sharp  distinguishing  point  and  therefore  proposes  to  drop  the  names  mono-, 
di-,  and  tricasein. 

Casein  solutions  do  not  coagulate  on  boiling,  but  solutions  of  casein- 
lime  are  covered,  like  milk,  with  a  pellicle.  They  are  precipitated  by  very 
little  acid,  but  the  presence  of  neutral  salts  retards  the  precipitation.  A 
casein  solution  containing  salt  or  ordinars^  milk  requires,  therefore,  more 
acid  for  precipitation  than  a  salt-free  solution  of  casein  of  the  same  concen- 
tration. The  precipitated  casein  dissolves  very  easily  again  m  a  small 
excess  of  hydrochloric  acid,  but  less  easily  in  an  excess  of  acetic  acid.  The 
combination  between  casein  and  acid,  and  especially  the  combination  with 
lactic  acid,  which  has  been  carefully  studied  by  Laxa,-*  are,  like  other 
protein  and  acid  compounds,  precipitated  by  neutral  salts.  Tliese  acid 
solutions  are  precipitated  by  mineral  acids  in  excess.     Casein  is  precipitated 

'  Laqueur  and  Sackur,  Hofmeister's  Beitrage,  3;  M.  Arthus,  Theses  presentees 
a  la  faculte  des  sciences  de  Paris,  1893. 

-  Soldner,  Die  Salze  aer  MUch,  etc.;  Couraut,  1.  c.  In  regard  to  the  salts  of  casein 
see  the  investigations  of  Soldner,  Maly's  Jahresber.,  25,  and  J.  Rohmann,  Berlin,  klin. 
Wochenschr. ,  1895.     See  also  Raudnitz,  Ergebnisse  der  Physiol.,  2,  Abt.  1. 

^  Hofmeister's  Beitrage,  7. 

'  Milchwirtsch.  Centralbl.,  1905,  Heft  12. 


520  MILK. 

from  neutral  solutions  or  from  milk  by  common  salt  containing  calcium  or 
magnesium  sulphate  in  substance,  without  changing  its  properties.  Metallic 
salts,  such  as  alum,  zinc  sulphate,  and  copper  sulphate,  completely  pre- 
cipitate the  casein  from  neutral  solutions. 

On  drying  at  100°  C,  casein,  according  to  Laqueur  and  Sackur,  decom- 
poses and  splits  into  two  bodies.  One  of  these,  called  caseid,  is  insoluble 
in  dilute  alkalies,  while  the  other,  the  isocasein,  is  soluble  therein.  The 
isocasein  is  a  stronger  acid  and  has  other  precipitation  limits  and  a  some- 
what lower  equivalent  weight  than  the  casein. 

The  property  which  is  the  most  characteristic  of  casein  is  that  it  coagu- 
lates with  rennet  in  the  presence  of  a  sufficiently  great  amount  of  lime-salts. 
In  solutions  free  from  lime-salts  the  casein  does  not  coagulate  with  rennet, 
but  it  is  changed  so  that  the  solution  (even  if  the  enzyme  is  destroyed  by 
heating)  yields  a  coagulated  mass,  having  the  properties  of  a  curd,  if  lime- 
salts  are  added.  The  rennet  enzyme,  rennin,  has  therefore  an  action  on 
casein  even  in  the  absence  of  lime-salts.  These  last  are  only  necessary 
for  the  coagulation  or  the  separation  of  the  curd,  and  the  process  of  coagu- 
lation is  hence  a  two-phase  process.  The  first  phase  is  the  transformation 
of  the  casein  by  the  rennin,  the  second  is  the  visible  coagulation  caused  by 
the  lime-salts.  This  fact,  which  was  first  proved  by  Hammarsten,  was 
later  confirmed  by  Arthus  and  Pages  and  recently  closely  studied  by 
FuLD,  Spiro,  and  Laqueur.^ 

The  curd  formed  on  the  coagulation  of  milk  contains  large  quantities  of 
calcium  phosphate.  According  to  Soxhlet  and  Soldner,  the  soluble 
lime-salts  are  of  essential  importance  only  in  coagulation,  while  the  calcium 
phosphate  is  without  importance.  According  to  Courant,  the  calcium- 
casein  on  coagulation  may  carry  down  with  it,  if  the  solution  contains 
dicalcium  phosphate,  a  part  of  this  as  tricalcium  phosphate,  leaving  mono- 
calcium  phosphate  in  the  solution.  A  solution  of  calcium-casein  is  not 
coagulated  by  rennin  alone  but  only  when  soluble  lime-salts  are  added. 
Milk  or  casein  solutions  may  indeed  be  precipitated  without  rennin  by  the 
addition  of  a  sufficiently  large  amount  of  calcium  chloride.  We  are  not 
quite  clear  as  to  the  importance  of  the  lime-salts  for  the  rennin  coagulation, 
and  the  views  are  still  somewhat  variable  on  this  question.  The  same  is 
true  for  the  chemical  processes  going  on  in  rennin  coagulation.  If  one 
makes  use  of  a  pure  solution  of  casein  and  as  pure  rennin  as  possible,  then 
after  coagulation  it   is  always  found  that  the  filtrate  contains  very  small 

^  See  Maly's  Jahresber.,  2  and  4;  also  Hammarsten,  Zur  Kenntnis  des  Kaseins  und 
der  Wirkung  des  Labfermentes,  Nova  Acta  Reg.  Soc.  Scient.  Upsala,  1877,  Fest- 
schrift; Zeitschr.  f.  physiol.  Chem.,  22;  Arthus  et  Pages,  Arch,  de  Physiol.  (5),  2,  and 
M^m.  Soc.  biol.,  43;  Fuld,  Hofmeister's  Beitrage,  2,  and  Ergebnisse  der  Physiol.,  1, 
Abt.  1,  where  a  good  review  of  the  literature  may  be  found;  Spiro,  Hofmeister's 
Beitrage,  fi  and  7,  with  Reichel,  ibid.,  7  and  8;  Laqueur,  ibid.,  7. 


CASEIN.  521 

amounts  of  a  proteid,  the  whey-proteid,  which  has  other  properties  and  a 
lower  content  of  nitrogen  (13.2  per  cent  N,  Koster  ^)  than  the  casein. 
The  chief  portion  of  the  casein,  sometimes  given  as  more  than  90  per  cent, 
separates  on  coagulation  as  a  body,  the  paracasein  (or  curd),  which  is 
closely  related  to  casein.  The  question  whether  a  cleavage  of  the  casein 
takes  place  here  is  still  unsettled.  The  paracasein  2  is  not  further  changed 
by  the  rennet  enzyme ;  it  is  much  more  readily  precipitated  by  CaCl2  than 
a  casein  solution  of  the  same  concentration,  and  the  precipitation  limits  for 
saturated  ammonium- sulphate  solution,  the  upper  as  well  as  the  lower 
limit,  lie.  according  to  Laqueur,  lower  with  paracasein  than  with  casein. 
The  internal  friction  of  paracasein  solutions  is  also,  according  to  him,  less 
than  that  of  the  casein  solutions  and  indeed  even  to  20  per  cent. 

In  the  processes  of  rennet  coagulation  we  may,  as  Reichel  and  Spiro 
have  sho'VMi,  have  a  diminution  of  the  action  by  an  apparent  consumption 
of  the  ferment.  This  diminution  is  indeed,  as  the  above  investigators 
found,  not  caused  by  the  process  of  rennet  coagulation  and  is  therefore 
not  to  be  considered  as  a  consumption  of  the  enzyme.  It  depends  upon  a 
division  of  the  rennin  between  the  curd  and  the  whey  taking  place  accord- 
ing to  a  constant  factor. 

Fresh,  unchanged  milk  does  not,  as  is  kno'mi,  coagulate  on  boiling ;  but  in 
not  too  rapid  action  of  rennin  a  state  may  be  observed  in  which  the  milk  coagu- 
lates on  heating  (metacasein  reaction).  A  solution  of  paracasein  lactate,  accord- 
ing to  Laxa,  coagulates  with  rennin  the  same  as  a  solution  of  casein  lactate, 
which  indicates,  according  to  Laxa,  that  the  paracasein  is  transformed  into  casein 
again  by  the  lactic  acid.  But  as  a  precipitation  of  the  paracasein  from  the  acid 
solution  is  perhaps  a  pepsin  action,  the  transformation  of  the  paracasein  into 
casein  by  the  lactic  acid  must  not  be  considered  as  proved.  As  the  commercial 
rennet  extracts  may  contain  also  other  enzymes  besides  rennin,  the  formation  of 
proteoses  in  rennin  coagulation,  as  observed  by  E.  Petry,'  must  not  be  con- 
sidered as  a  rennin  action  without  further  study. 

In  the  digestion  of  casein  mth  pepsin-hydrochloric  acid  primarily  a 
phosphorized  proteose  is  formed,  from  which  then  the  pseudonuclein  is 
split  off  (Salkowski).  The  quantity  thus  split  off  is  verj^  variable,  as 
shown  by  the  researches  of  Salkowski,  Hahx,  Moraczewski,  Sebeliex,  and 
Zaitschek."*    The  amount  of  phosphorus  in  the  pseud onucleins  obtained  also 

■  See  Maly's  Jahresber.,  11. 

^  It  has  been  recently  proposed  to  de.signate  the  ordinary  ca.sein  as  caseinogen  and 
the  curd  as  casein.  Although  such  a  propcsition  is  theoretically  correct,  it  leads  in 
practice  to  confusion.  On  this  account  the  author  calls  the  curd  paracasein,  according 
to  Schulze  and  Rose  (Landwirthsch.  Versuchsstat.,  31).  A  sununary  of  the  literature 
on  the  casein  coagulation  may  be  found  in  E.  Fuld,  Ergebnisse  der  Physiol.,  1;  Raud- 
nitz,  ibicl.,  2;   and  Laqueur,  Biochem.  Centralbl.,  4,  344. 

'Laxa,  I.e.;  Petry,  Wien.  klin.  Wochenschr.,  1906. 

^Salkowski,  Zeitschr.  f.  physiol.  Chem.,  2";  Salkowski  and  Hahn,  Pfliigcr's  Arch., 
59;  Salkowski,  ibuL,  63;  v.  Moraczewski,  Zeitschr.  f.  physiol.  Chem.,  20;  Sebelien, 
ibid..  20;    Zaitschek,  Pfliiger's  Arch.,  104. 


522  MILK. 

varies  considerably.  According  to  Salkowski  the  quantity  of  pseudo- 
nuclein  split  off  is  dependent  upon  the  relationship  between  the  casein  and 
the  digestion  fluid,  e.g.,  the  quantity  of  the  pseudonucleins  diminishes  as 
the  pepsin-hydrochloric  acid  increases.  In  the  presence  of  5UU  grams  of 
pepsin-hydrochloric  acid  to  1  gram  of  casein  Salkowski  digested  the  latter 
completely  without  obtaining  any  pseudonuclein. 

In  peptic  as  well  as  tryptic  digestion  a  part  of  the  organic  phosphorus 
is  split  off  as  orthophosphoric  acid,  the  quantity  increasing  as  the  digestion 
progresses.  Another  part  of  the  phosphorus  is  retained  in  organic  com- 
bination in  the  proteoses  as  well  as  in  the  true  peptones  (Salkowski,  Biffi, 
Alexander!). 

From  the  products  of  peptic  digestion  of  casein,  after  the  separation  of  the 
pseudonuclein,  Salkowski  ^  has  isolated  an  acid  rich  in  phosphorus.  He  considers 
this  a  ■paranucleic  acid.  It  is  soluble  in  water,  insoluble  in  alcohol,  levorotatory, 
and  has  the  following  composition:  C  42.51-42.96,  H  6.97-7.09,  N  13.25-13.55, 
and  P  4.05-4.31  per  cent.  The  acid  differs  from  the  nucleic  acids  in  that  it  gives 
the  biuret  test  and  a  faint  xanthoproteic  reaction.  Presupposing  its  puiity,  it 
is  not  an  acid  comparable  to  the  nucleic  acids. 

Casein  may  be  prepared  in  the  following  way:  The  milk  is  diluted  with 
4  vols,  of  water  and  the  mixture  treated  with  acetic  acid  to  0.75 — 1  p.  m. 
Casein  thus  obtained  is  purified  by  repeatedly  dissolving  in  water  with  the 
aid  of  the  smallest  quantity  of  alkali  possible,  by  filtering  and  reprecipi- 
tating  with  acetic  acid  and  thoroughly  washing  with  water.  Most  of  the 
milk-fat  is  retained  by  the  filter  on  the  first  filtration,  and  the  casein  con- 
taminated with  traces  of  fat  is  purified  by  treating  with  alcohol  and  ether. 

Lactoglobulin  was  obtained  by  Sebelien  from  cow's  milk  by  saturating 
it  with  NaCl  in  substance  (which  precipitated  the  casein)  and  saturating 
the  filtrate  with  magnesium  sulphate.  As  far  as  it  has  been  investigated 
it  had  the  properties  of  serglobulin;  the  globulin  isolated  by  Tiemann^ 
from  colostrum  had  nevertheless  a  markedly  low  content  of  carbon,  namely, 
49.83  per  cent. 

Lactalbumin  was  first  prepared  in  a  pure  state  from  milk  by  Sebelien. 
Its  composition  is,  according  to  him,  C  52.19,  H  7.18,  N  15.77,  S  1.73, 
O  23.13  per  cent.  Lactalbumin  has  the  properties  of  the  albumins,  and  it 
crystaUizes  according  to  Wichmann  ^  in  forms  similar  to  ser-  or  ovalbumin. 
It  coagulates,  according  to  the  concentration  and  the  amount  of  salt  in 
solution,  at  72-84°  C.  It  is  similar  to  seralbumin,  but  differs  from  it  in 
having  a  considerably  lower  specific  rotatory  power:    (a)D=  —  37°. 

'Salkowski,  1.  c;  Biffi,  Virchovv's  Arch.,  152;  Alexander,  Zeitschr.  f.  physiol. 
Chem.,  25. 

^  Zeitschr.  f.  physiol.  Chem.,  32. 

s/6w/.,25. 

*  Sebelien,  Zeitschr.  f.  physiol.  Chem.,  9;  Wichmann,  ibid.,  27. 


LACTALBUMIX    AXD    ENZYMES.  523 

The  principle  of  the  preparation  of  lact  albumin  is  the  same  as  for  the 
preparation  of  seralbumin  from  serum.  The  casein  and  the  globulin  are 
removed  by  MgS04  in  substance  and  the  filtrate  treated  as  previously 
stated  (page  182). 

The  occurrence  of  other  proteins,  such  as  proteoses  and  peptones,  in  milk  has 
not  been  positively  proved.  These  bodies  are  easily  produced  as  laboratory 
products  from  the  other  proteins  of  the  milk.  Such  a  laboratorj^  product  is 
Millon's  and  Comaille's  lactoprotein,  which  is  a  mixture  of  a  little  casein  with 
changed  albimiin,  and  proteose  '  which  is  formed  by  chemical  action.  In  regard 
to  opalisin,  see  Human  ^lilk,  p.  531. 

Milk  also  contains,  according  to  Siegfried,^  a  nucleon  related  to  phos- 
phocarnic  acid,  and  which  yields  fermentation  lactic  acid  (instead  of  para- 
^actic  acid)  and  a  special  carnic  acid,  orylic  acid  (instead  of  muscle  camic 
acid),  as  cleavage  products.  Lactophosphocamic  acid  may  be  precipitated 
as  an  iron  compound  from  the  milk  freed  from  casein  and  coagulable 
proteins  as  well  as  from  earthy  phosphates. 

Milk  also  contains  enzymes  of  various  kinds.  Of  these  we  must  mention 
catalase,  oxidases,  peroxidases,  and  reductases,  but  the  statements  as  to  their 
occurrence  in  the  milk  from  different  animals  are  not  unanimous.  An 
amylolytic  enzyme  w^hich  converts  starch  into  maltose  occurs  especially  in 
human  milk,  while  it  is  absent  in  cow's  milk  or  occurs  only  to  a  slight  extent. 
A  fermentation  enzyme  which  in  the  absence  of  micro-organisms  deeom- 
poses  the  lactose  into  lactic  acid,  alcohol,  and  CO2.  occurs,  according  to 
Stoklasa^  and  his  co-workers,  in  cow's  milk  as  well  as  in  human  milk. 
Human  milk,  as  well  as  cow's  milk,  contains  a  lipase  which  has  the  prop- 
erty at  least  of  acting  upon  monobutyrin.  Babcock  and  Russel  have 
found  in  these  two  kinds  of  milk,  as  well  as  certain  others,  a  proteolytic 
enzyme  which  they  call  galactase  and  which  is  allied  to  trypsin,  but  differs 
therefrom  in  that  it  develops  ammonia  from  milk  even  in  the  early  stages 
of  digestion.  The  occurrence  of  such  an  enzyme  is  denied  by  Z.\itschek 
and  V.  SzoNTAGH,  but  on  the  other  hand  Vaxdevelde,  de  Waele,  and 
Sugg  *  confirm  the  occurrence  of  a  proteolytic  enzyme  in  milk. 

Orotic  acid,  C,5HiiX204.2H20,  is  the  name  given  by  Biscaro  and  Belloxi  ^ 
to  a  new  constituent  of  milk  which  they  have  discovered.  This  acid,  which  can 
be  precipitated  by  basic  lead  acetate  from  whey  free  from  protein,  is  slightly 
soluble  in  water,  crystalline,  and  gives  several  crystalline  salts.  The  mono- 
methyl  and  ethyl  esters  of  this  acid  are  also  knoma.  It  j-ields  urea  on  treatment 
with  potassium  permanganate. 

'  See  Hammar.«ten.  Maly's  Jahresber. ,  6,  13. 

^  Zeitschr.  f.  phy.siol.  Chem.,  21  and  22. 

3  See  Chem.  Centralhl.,  190.5,  1,  107. 

^  Babcock  and  Russel,  Centralhl.  f.  Bakt.  u.  Parisitenkunde  (II),  6,  and  Maly's 
Jahresber.,  31;  Zaitschek  and  v.  Szontagh,  Pflijger's  Arch.,  104;  Vandevelde  de 
Waele,  and  Sugg,  Hofmeister's  Beitrage,o. 

5  See  Chem.  Centralbl.,  1905,  2,  6-3. 


524  MILK. 

Lactose,  milk-sugar,  C12H22O11  +  H2O.  This  sugar,  on  hydrolysis,  can 
be  split  into  two  hexoses,  dextrose  and  galactose.  It  yields  mucic  acid, 
besides  other  organic  acids,  by  the  action  of  dilute  nitric  acid.  LevuUnic 
acid  is  formed,  besides  formic  acid  and  humin  substances,  by  the  stronger 
action  of  acids.  By  the  action  of  alkalies,  amongst  other  products  we  find 
lactic  acid  and  pyrocatechin. 

Milk-sugar  occurs,  as  a  rule,  only  in  milk,  but  it  has  also  been  found  in 
the  urine  of  pregnant  women  on  stagnation  of  milk,  as  well  as  in  the  urine 
after  partaking  of  large  quantities  of  the  same  sugar. 

Lactose,  of  which,  according  to  Tanret,i  there  are  three  modifica- 
tions, occurs  ordinarily  as  colorless  rhombic  crystals  with  1  molecule  of 
water  of  crystallization,  which  is  driven  off  by  slowly  heating  to  100°  C, 
but  more  easily  at  130-140°  C.  At  170°  to  180°  C.  it  is  converted  into  a 
brown  amorphous  mass,  lactocaramel,  CgHioOs.  On  quickly  boiling  down 
a  milk-sugar  solution,  anhydrous  milk-sugar  separates  out.  Milk-sugar 
dissolves  in  6  parts  cold  or  in  2.5  parts  boiling  water;  it  has  a  faintly  sweet- 
ish taste.  It  does  not  dissolve  in  ether  or  absolute  alcohol.  Its  solutions 
are  dextrogyrate.  The  rotatory  power,  which  on  heating  the  solution  to 
100°  C.  becomes  constant,  is  (q;)d= +52.5°.  Milk-sugar  combines  with 
bases;   the  alkali  combinations  are  insoluble  in  alcohol. 

Milk-sugar  is  not  fermentable  with  pure  yeast.  It  undergoes,  on  the 
contrary,  alcoholic  fermentation  by  the  action  of  certain  schizomycetes,  and 
according  to  E.  Fischer  2  the  milk-sugar  is  first  split  into  dextrose  and 
galactose  by  an  enzyme,  lactase,  existing  in  the  fungus.  The  preparation 
of  milk-wine,  "kumyss,"  from  mare's  milk  and  "kephir^'  from  cow's  milk  is 
based  upon  this  fact.  Other  micro-organisms  also  take  part  in  this  change, 
causing  a  lactic-acid  fermentation  of  the  milk-sugar. 

Lactose  responds  to  the  reactions  of  dextrose,  such  as  Moore's, 
Trommer's,  and  Rubner's,  and  the  bismuth  test.  It  also  reduces  mer- 
curic oxide  in  alkaline  solutions.  After  warming  with  phenylhydrazine 
acetate  it  gives  on  cooling  a  yellow  crystalline  precipitate  of  phenyl- 
lactosazone,  C24H32N4O9.  It  differs  from  cane-sugar  by  giving  positive 
reactions  with  Moore's  or  Trommer's  and  the  bismuth  test,  and  also  in 
that  it  does  not  darken  when  heated  with  anhydrous  oxalic  acid  to  100°  C. 
It  differs  from  dextrose  and  maltose  by  its  solubility  and  crystalline  form, 
but  especialh^  by  its  not  fermenting  A\ith  yeast  and  by  yielding  mucic 
acid  with  nitric  acid. 

The  osazone  obtained  with  phenylhydrazine  acetate,  which  melts  at  200° 
C,  differs  from  the  other  osazones  by  being  inactive  when  0.2  gram  is  dis- 
solved in  4  c.c.  of  pyridine  and  6  c.c.  of  absolute  alcohol  and  viewed  through 
a  layer  10  centimetres  long  (Neuberg^). 

»  Bull.  Soc.  chim.  (.3),  13.         =  Ber.  d.  d.  chem.  Gesellsch.,  27.         » Ibid.,  32. 


ANALYSIS  OF   MILK.  525 

For  the  preparation  of  milk-sugar  we  make  use  of  the  by-product  in 
the  preparation  of  cheese,  the  sweet  whey.  The  protein  is  removed  by 
coagulation  with  heat,  and  the  fiUrate  evaporated  to  a  syrup.  The  cr>-stals 
which  separate  after  a  certain  time  are  recr^-staUized  from  water  after 
decolorizing  with  animal  charcoal.  A  pure  preparation  may  be  obtained 
from  the  commercial  milk-sugar  by  repeated  recrystallization.  The  C[uan- 
titative  estimation  of  milk-sugar  may  be  performed  either  by  the  polar- 
istrobometer  or  by  means  of  titration  with  Fehlixg's  solution.  Ten  c.c. 
of  Fehlixg's  solution  correspond  to  0.0676  gram  of  milk-sugar  in  0.5-1.5 
per  cent' solution  after  boiling  for  six  minutes.  (In  regard  to  Fehlixg's 
solution  and  the  titration  of  sugar  see  Chapter  XV.) 

RiTTHAUSEN  has  found  another  carbohydrate  in  milk  which  is  soluble  in  water, 
non-crystallizable,  which  has  a  faint  reducing  action,  and  which  yields  on  boiling 
with  an  acid  a  body  having  a  greater  reducing  power.  Laxdwehr  considers 
this  as  anunal  gum,  and  Bechamp  ^  as  dextrin. 

The  mineral  bodies  of  milk  will  be  treated  in  connection  ^\ith  its  quan- 
titative composition. 

The  methods  for  the  quantitative  analysis  of  milk  are  verj-  numerous, 
and  as  they  cannot  all  be  treated  here,  we  will  give  the  chief  points  of  a 
few  of  the  methods  considered  most  trustworthy  and  most  frequently 
employed. 

In  determining  the  solids  a  carefully  weighed  quantity  of  milk  is  mixed 
with  an  equal  weight  of  heated  quartz  sand,  fine  glass  powder,  or  asbestos. 
The  evaporation  is  first  done  on  the  water-bath  and  finished  in  a  current 
of  carbon  dioxide  or  hydrogen  not  above  100°  C. 

The  inineral  bodies  are  determined  by  incinerating  the  milk,  using  the 
precautions  mentioned  in  the  text-books.  The  results  obtained  for  the 
phosphoric  acid  are  incorrect  on  accoimt  of  the  burning  of  phosphorized 
bodies,  such  as  casein  and  lecithin.  We  must  therefore,  according  to  Sold- 
NER,  subtract  in  round  numbers  25  per  cent  from  the  total  phosphoric  acid 
found  in  the  milk.  The  quantity  of  sulphate  in  the  ash  also  depends  on 
the  combustion  of  the  proteins. 

In  the  determination  of  the  total  amount  of  proteins  Ritthausex's 
method  is  employed,  namely,  the  precipitation  of  the  milk  ^^•ith  copper  sul- 
phate according  to  the  modification  suggested  by  Muxk.^  He  precipitates 
all  the  proteins  by  means  of  cupric  hydrate  at  boiling  heat,  and  determines 
the  nitrogen  in  "the  precipitate  by  means  of  Kjeldahl's  method.  This 
modification  gives  more  exact  results. 

The  older  method  of  Puls  and  Stexberg,  in  which  the  precipitant  is 
alcohol,  is  too  complicated  and  not  sufficiently  reliable.  Sebeliex  has  sug- 
gested a  very  good  method.  Three  to  four  grams  of  milk  are  diluted  -^ith 
an  equal  volume  of  water,  a  little  common-salt  solution  added,  and  the  pro- 
teins precipitated  with  an  excess  of  tannic  acid.  The  precipitate  is  washed 
with  cold  water,  and  then  the  quantity  of  nitrogen  determined  by  Kjeldahl's 
method.  The  total  nitrogen  foimd  when  multiplied  by  6.37  (casein  and 
lactalbumin  contain  both  15.7  per  cent  nitrogen)  gives  the  total  quantity 

'  Ritthausen,  Journ.  f.  prakt.  Chem.  (X.  F.),  15;  Landwehr,  foot-note  1,  p.  67; 
Bechamp,  Bull.  Sec.  chim.  (3)    6. 

'  Ritthausen,  1   c;   I.  Munk,  Virchow's  Arch.,  134. 


526  MILK. 

of  proteins.  This  method,  which  is  readily  performed,  gives  very  good 
results.  I.  MuxK  used  this  method  in  the  analysis  of  woman's  milk.  In 
this  case  the  quantity  of  nitrogen  found  must  be  multiplied  by  6.34.  G. 
SiMOX  1  has  found  that  the  precipitation  •s\-ith  tannic  acid,  also  \\ith  phos- 
photungstic  acid,  is  the  simplest  and  most  accurate.  The  objection  to 
this  and  other  methods  in  which  the  proteins  are  precipitated  is  that  per- 
haps other  bodies  (extractives)  may  be  carried  down  at  the  same  time  (Cam- 
ERER  and  Soldxer2).     It  is  not  kno'^n  to  '^hat  extent  this  takes  place. 

A  part  of  the  nitrogen  in  the  milk  exists  as  extractives,  and  this  nitrogen  is 
calculated  as  the  difference  between  the  total  nitrogen  and  the  protein  nitrogen. 
According  to  Muxk's  anal3-ses  about  i^  of  the  total  nitrogen  belongs  to  the 
extractives  in  cow's  milk,  and  tx  in  woman's  milk.  Camerer  and  Soldner 
determine  the  nitrogen  in  the  filtrate  from  the  tannic-acid  precipitate  by  Kjel- 
dahl's  method,  and  also  according  to  Hufner's  method  (hypobromite).  In  this 
way  they  found  18  milligrams  of  nitrogen  accordmg  to  HiJFNER  (urea,  etc.)  in  100 
grams  of  cow's  milk. 

To  determine  the  casein  and  albumin  separately  we  may  make  use  of 
the  method  first  suggested  by  Hoppe-Seyler  and  Tolmatscheff,^  in 
which  the  casein  is  precipitated  by  magnesium  sulphate.  According  to 
Sebelien  the  milk  is  diluted  ^\ith  its  o^\'n  volume  of  a  saturated  mag- 
nesium-sulphate solution,  then  saturated  with  the  salt  in  substance,  and 
the  precipitate  then  filtered  and  washed  vAih.  a  saturated  magnesium- 
sulphate  solution.  The  nitrogen  is  determined  in  the  precipitate  by  Kjel- 
dahl's  method,  and  the  quantity  of  casein  (  +  giobulin)  determined  by 
multiplying  the  result  by  6.37.  The  quantity  of  lactalbumin  may  be 
calculated  as  the  difference  between  the  casein  and  the  total  proteins 
found.  The  lactalbumin  may  also  be  precipitated  by  tannic  acid  from  the 
filtrate  from  the  casein  precipitate  containing  MgS04,  after  diluting  with 
water,  the  nitrogen  determined  by  Kjeldahl's  method  and  the  result  mul- 
tiplied by  6.37. 

Schlossmaxx^  suggests  an  alum  solution,  which  precipitates  the  casein, 
in  order  to  separate  the  casein  from  the  other  proteins,  the  albumin  can 
be  precipitated  from  the  filtrate  by  tannic  acid.  The  nitrogen  in  the  pre- 
cipitate is  determined  b}^  the  Kjeldahl's  method.  This  method  has 
recently  been  tested  by  Simox  and  he  recommends  it  highly. 

The  fat  is  gravimetrically  determined  by  thoroughly  extracting  the 
dried  milk  with  ether,  evaporating  the  ether  from  the  extract,  and  weighing 
the  residue.  The  fat  may  be  determined  by  aerometric  means  by  adding 
alkali  to  the  milk,  shaking  with  ether,  and  determining  the  specific  gravity 
of  the  fat  solution  by  means  of  Soxhlet's  apparatus.  In  determining  the 
amount  of  fat  in  a  large  number  of  samples  the  lactocrit  of  De  Laval  may 
be  used  \v\ih  success.  The  milk  is  first  mixed  \\i\h  an  equal  volume  of  a 
mixture  of  glacial  acetic  and  concentrated  sulphuric  acid,  warmed  7-8 
minutes  on  the  water-bath,  and  the  mixture  poured  in  graduated  tubes, 
which  are  placed  in  the  centrifugal  machine  at  50°  C.     The  height  of  the 

'Puis,  Pfliisier's  Arch.,  13;    Stenberg,  IVIaly's  Jahresber.,  7;   Sebelien,  Zeitschr.  f. 
physiol.  Chem.,  13;    Simon,  ibid.,  33. 
2  Zeitschr.  f .  Biologie,  33  and  3fi. 
^  Hoppc-Seyler,  Med.  chem.  Untersuch.,  272. 
*  Zeitschr.  f.  physiol.  Chem.,  22. 


QUANTITATIVE  COMPOSITIOX   OP    COW'S  MILK.  527 

layer  of  fat  gives  its  quantity.  The  numerous  and  verj-  exact  analyses  of 
NiLSOX  ^  have  shown  that  \\ith  milks  containing  small  quantities  of  fat, 
below  1.5  per  cent,  the  older  corrections  are  unnecessary,  and  that  this 
method  gives  excellent  results  if  we  use  lactic  acid  treated  with  5  per  cent 
hydrochloric  acid  instead  of  the  above  mixture  of  glacial  acetic  acid  and 
sulphuric  acid.  There  are  numerous  other  methods  for  estimating  milk- 
fat  but  they  cannot  be  considered  here. 

In  determining  the  milk-sugar  the  proteins  are  first  removed.  For 
this  purpose  we  precipitate  either  ^^■ith  alcohol,  which  must  be  evaporated 
from  the  filtrate,  or  by  diluting  with  water,  and  remo\ing  the  casein  by 
the  addition  of  a  little  acid,  and  the  lactalbumin  by  coagulation  at  boiling 
heat.  The  sugar  is  determined  by  titration  with  Fehlixg's  or  Knapp's 
solution  (see  Chapter  X\).  The  principle  of  the  titration  is  the  same  as 
for  the  titration  of  sugar  in  the  urine:  10  c.c.  of  Fehlixg's  solution  corre- 
spond to  0.0676  gram  of  milk-sugar;  10  c.c.  of  Kxapp's  solution  correspond 
to  0.0311-0.0310  gram  of  milk-sugar,  when  the  saccharine  liquid  contains 
about  i-1  per  cent  of  sugar.  In  regard  to  the  modus  operandi  of  the  titra- 
tion we  must  refer  the  reader  to  more  complete  works  and  to  Chapter  XV, 

Instead  of  these  volumetric  determinations  other  methods  of  estima- 
tion, such  as  Allihx's  method,  the  polariscope  method,  and  others,  may 
be  used.  In  calculating  the  analysis  or  in  determining  the  soUds  it  is  of 
importance  to  remember,  as  suggested  by  Caterer  and  Soldxer,  that  the 
milk-sugar  in  the  residue  is  anhydrous.  Many  other  methods  for  deter- 
mining the  milk-sugar  have  been  suggested  and  recommended. 

The  quantitative  composition  of  cow's  milk  is  naturally  xevy  variable. 
The  average  obtained  by  Koxig-  is  as  follows  in  1000  parts: 

Water.  Solids.  Casein.         Albumin.  Fats.  Sugar.  Salts. 

871.7  128. .3  30.2  .5.3  36.9  48.8  7.1 


35.5 

The  quantity  of  mineral  bodies  in  1000  parts  of  cow's  milk  is,  according 
to  the  analyses  of  Soldxer,  as  follows:  K2O  1.72,  Na20  0.51,  CaO  1.98, 
MgO  0.20,  P2O5  1.82  (after  correction  for  the  pseud onuclein),  CI  0.98  grams. 
Buxge3  foimd  0.0035  gram  Fe203.  According  to  Soldxer  the  K,  Na, 
and  CI  are  foimd  in  the  same  quantities  in  ^^'hole  milk  as  in  milk-serum. 
Of  the  total  phosphoric  acid  36-56  per  cent  and  of  the  lime  53-72  per  cent 
is  not  in  simple  solution.  A  part  of  this  lime  is  combined  -uith  the  casein; 
the  remainder  is  found  united  with  the  phosphoric  acid  as  a  mixture  of 
dicalcium  and  tricalcium  phosphates  which  is  kept  dissolved  or  suspended  by 
the  casein.  The  bases  are  in  excess  of  the  mineral  acids  in  the  milk-serum. 
The  excess  of  the  first  is  combined  with  organic  acids,  which  correspond 
to  2.5  p.  m.  citric  acid  (Soldxer). 

The  gases  of  the  milk  consist  chiefly  of  CO2.  besides  a  little  X  and 


*  See  Maly's  Jahresber.,  21. 

'  Chemie  der  menschlichen  Xahrungs-  unci  Genussmittel,  4.  Aufl. 

3  Zeitschr.  f.  Biologie,  10. 


528  MILK. 

traces  of  O.  Pfluger  i  found  10  vols,  per  cent  CO2  and  0.6  vol.  per  cent 
N  calculated  at  0°  C.  and  760  mm.  pressure. 

The  variation  in  the  composition  of  cow's  milk  depends  on  several 
circumstances. 

The  colostrum,  or  the  milk  which  is  secreted  before  cahdng  and  in  the 
first  few  days  after,  is  yellowish,  sometimes  alkaline,  but  often  acid,  of 
higher  specific  gravity,  1.046-1.080,  and  richer  in  solids  than  ordinary 
milk.  The  colostrum  contains,  besides  fat-globules,  an  abundance  of 
colostrum-corpuscles — nucleated  granular  cells  0.005-0.025  mm.  in  diam- 
eter with  abundant  fat-granules  and  fat-globules.  The  fat  of  colostrum 
has  a  somewhat  higher  melting-point  and  is  poorer  in  volatile  fatty  acids 
than  the  fat  from  ordinary  milk  (Nilson^).  The  iodine  equivalent  of  the 
colostrum-fat  is  higher  than  that  of  milk-fat.  The  quantity  of  cholesterin 
and  lecithin  is  generally  greater.  The  most  apparent  difference  between 
it  and  ordinary  milk  is  that  colostrum  coagulates  on  heating  to  boiling 
because  of  the  absolutely  and  relatively  greater  quantities  of  globulin  and 
albumin  that  it  contains.^  The  composition  of  colostrum  is  very  variable. 
KoNiG  gives  as  average  the  following  figures  in  1000  parts: 

Water.  Solids.  Casein.     Albumin  and  Globulin.      Fat.  Sugar.  Salts. 

746.7  253.3  40.4  136.0  35.9  26.7  15.6 

The  influence  which  food  exercises  upon  the  composition  of  milk  will 
be  discussed  in  connection  with  the  chemistry  of  the  milk  secretion. 

In  the  following  table  is  given  the  average  composition  of  skimmed  milk  and 
certain  other  preparations  of  milk: 

Water.  Proteins.  Fat.  Sugar.  Lactic  Acid.  Salts. 

Skimmed  milk 906.6  31.1  7.4  47.5          ...  7.4 

Cream 655.1  36.1  267.5  35.2          ...  6.1 

Buttermilk 902.7  40.6  9.3  37.3         3.4  6.7 

Whey 932.4  8.5  2.3  47.0         3.3  6.5 

KuMYSS  and  kephir  are  obtained,  as  above  stated,  by  the  alcoholic  and  lactic- 
acid  fermentation  of  the  milk-sugar,  the  former  from  mare's  milk  and  the  latter 
from  cow's  milk.  Large  quantities  of  carbon  dioxide  are  formed  thereby,  and 
besides  the  protein  bodies  of  the  milk  are  partly  converted  into  proteoses  and 
peptones,  which  increase  the  digestibility.  The  quantity  of  lactic  acid  in  these 
preparations  may  be  about  10-20  p.  m.  The  quantity  of  alcohol  varies  from  10  to 
35  p.  m. 

Milk  of  other  Animals.  Goat's  milk  has  a  more  yellowdsh  color  and  another, 
more  specific  odor  than  cow's  milk.  The  coagulum  obtained  by  acid  or  rennet 
is  more  solid  and  is  harder  than  that  from  cow's  milk.  Sheep's  milk  is  similar 
to  goat's  milk,  but  has  a  higher  specific  gravity  and  contains  a  greater  amount 
of  solids. 

'  Pfliiger's  Arch.,  2. 
'  Nilson,  1.  c. 

^See  Sebelien,  Maly's  Jahresber.,  18,  and  Tiemann,  Zeitschr.  f.  physiol.  Chem., 
25.     See  also  Simon,  ibid.,  33;   Winterstein  and  Strickler,  ibid.,  47. 


HUMAN    MILK.  529 

Mare's  milk  is  alkaline  and  contains  a  casein  which  is  not  precipitated  by 
acids  in  lumps  or  solid  masses,  but,  like  the  casein  from  woman's  milk,  in  fine 
flakes.  This  casein  is  only  incompletely  precipitated  by  rennet,  and  it  is  very 
similar  also  in  other  respects  to  the  casein  of  human  milk.  According  to  Beil  ' 
the  casein  from  mare's  and  cow's  milk  is  the  same,  and  the  different  behavior 
of  the  two  varieties  of  milk  is  due  to  different  amomits  of  salts  and  to  a  different 
relation  between  the  casein  and  the  albumin.  This  does  not  agree  with  the 
investigations  of  Zaitschek  and  v.  Szontagh,  who  find  that  the  casein  from 
mare's  milk,  like  that  from  human  and  ass's  milk,  is  digested  by  pepsin-hydro- 
chloric acid  without  leaving  a  residue.  The  milk  of  the  ass  is  claimed  by  older 
authorities  to  be  similar  to  human  milk,  but  Schlossmann  finds  it  considerably 
poorer  in  fat.  The  researches  of  Ellenberger  give  similar  results,  and  show 
great  similarity  between  ass's  milk  and  human  milk.  The  average  results  were 
15  p.  m.  protein  with  5.3  p.  m.  albumin  and  9.4  p.  m.  casein.  This  latter,  like 
human  casein,  does  not  yield  any  pseudonuclein  on  pepsin  digestion,  which  agrees 
well  with  the  above-mentioned  investigations  of  Zaitschek.  The  cjuantity  of 
nucleon  was  about  the  same  as  in  woman's  milk.  Thcf  Cjuantity  of  fat  was  15  p.  m., 
and  the  sugar  was  50-60  p.  m.  Reindeer  milk  is  characterized,  according  to 
Werenskiold,^  by  being  very  rich  in  fat,  144.6-197.3  p.  m.,  and  casein,  80.6-86.9 
p.  m. 

The  milk  of  carnivora  (the  bitch  and  cat)  is  acid  in  reaction  and  very  rich 
in  solids.  The  composition  of  the  milk  of  these  animals  varies  with  the  compo- 
sition of  the  food. 

To  illustrate  the  composition  of  the  milk  of  other  animals  the  following  figures, 
the  compilation  of  Konig,  are  given.  As  the  milk  of  each  kind  of  animal  may 
have  a  variable  composition,  these  figures  should  only  be  considered  as  examples 
of  the  composition  of  milk  of  various  kinds  :^ 

Milk  of  the  Water.  Solids.  Proteins.  Fat.  Sugar.  Salts. 

Dog 754.4  245.6  99.1  95.7  31.9  7.3 

Cat 816.3  183.7  90.8  33.3  49.1  5.8 

Goat 869.1  130.9  36.9  40.9  44.5  8.6 

Sheep 835.0  165.0  57.4  61.4  39.6  6.6 

Cow 871.7  128.3  35.5  36.9  48.8  7.1 

Horse 900.6  99.4  18.9  10.9  66.5  3.1 

Ass 900.0  100.0  21.0  13.0  63.0  3.0 

Pi? 823.7  167.3  60.9  64.4  40.4  10.6 

Elephant 678.5  321.5  30.9  195.7  88.4  6.5 

Dolphin 486.7  513.3         437.6  4.6 

Human  Milk. 

Woman's  milk  is  amphoteric  in  reaction.  According  to  Courant  its 
reaction  is  relatively  more  alkaline  than  cow's  milk,  but  it  has  nevertheless 
a  lower  absolute  reaction  for  alkalinity  as  well  as  for  acidity.  Courant 
found  between  the  tenth  day  and  the  fourteenth  month  after  confinement 
practically  constant  results.  The  alkalinity,  as  well  as  the  acidity,  was  a 
little  lower  than  in  childbed.  One  hundred  c.c.  of  the  milk  had  the  same 
average  alkalinity  as  10.8  c.c.  N/10  caustic  soda,  and  the  same  acidity 

'  Studien  iiber  die  Eiweissstoffe  des  Kumys  und  Kefirs,  St.  Petersburg,  1886 
(Ricker). 

^  Zaitschek,  1.  c;  Schlossmann,  Zeitschr.  f.  physiol.  Chem.,  22;  Ellenberger,  Arch, 
f.  (Anat.  u.)  Physiol.,  1899  and  1902;  Werenskiold,  Maly's  Jahresber.,  25. 

^  Details  in  regard  to  the  milk  of  different  animals  may  be  found  in  Proscher 
Zeitschr    f.  physiol,  Chem.,  24;    Ahderhalden,  ibid.,  27. 


530  MILK. 

as  3.6  c.c.  N/10  acid.  The  relationship  between  the  alkalinity  and  the 
acidity  in  woman's  milk  was  as  3:1,  and  in  cow's  milk  as  2.1:1.  The 
actual  reaction  determined  electrometrically  is,  according  to  Foa,i  still 
nearly  neutral,  like  the  other  kinds  of  milk. 

Human  milk  also  contains  fewer  fat-globules  than  cow's  milk,  but  they 
are  larger  in  size.  The  specific  gravity  of  woman's  milk  varies  between 
1.026  and  1.036,  generally  between  1.028  and  1.034.  It  is  highest  in  well- 
fed  and  lowest  in  poorly  fed  women.  The  freezing-point  is  lowered  on 
an  average  0.589°  C,  according  to  Winter  and  Parmentier^  constant  at 
0.55°,  and  the  molecular  concentration  is  0.318. 

The  fat  of  woman's  milk  has  been  investigated  by  Ruppel.  It  forms  a 
yellowish-white  mass,  similar  to  ordinary  butter,  having  a  specific  gravity 
of  0.966  at  15°  C.  It  melts  at  34.0°  C.  and  solidifies  at  20.2°  C.  The  fol- 
lowing fatty  acids  can  be  obtained  from  the  fat,  namely,  butyric,  caproic, 
capric,  myristic,  palmitic,  stearic,  and  oleic  acids.  The  fat  from  woman's 
milk  is,  according  to  Ruppel  and  Laves,^  relatively  poor  in  volatile  fatty 
acids.  The  non-volatile  fatty  acids  consist  of  one-half  oleic  acid,  while 
among  the  solid  fatty  acids  myristic  and  palmitic  acids  are  found  to  a 
greater  extent  than  stearic  acid. 

The  essential  qualitative  difference  between  woman's  and  cow's  milk 
seems  to  lie  in  the  jDroteins  or  in  the  more  accurately  determined  casein. 
A  number  of  older  and  younger  investigators'*  claim  that  the  casein  from 
woman's  milk  has  other  properties  than  that  from  cow's  milk.  The  essen- 
tial differences  are  the  following:  The  casein  from  woman's  milk  is  pre- 
cipitated with  greater  difficulty  with  acids  or  salts;  it  does  not  coagulate 
uniformly  in  the  milk  after  the  addition  of  rennet;  it  maybe  precipitated 
by  gastric  juice,  but  dissolves  completely  and  easily  in  an  excess  of  the 
same;  the  casein  precipitate  produced  by  an  acid  is  more  easily  soluble  in 
an  excess  of  the  acid ;  and  lastly,  the  clot  formed  from  the  casein  of  woman's 
milk  does  not  appear  in  such  large  and  coarse  masses  as  the  casein  from 
cow's  milk,  but  is  more  loose  and  flocculent.  This  last-mentioned  fact  is 
of  great  importance,  since  it  explains  the  generally  admitted  fact  of  the 
easy  digestibility  of  the  casein  from  woman's  milk.  We  are  not  clear  as 
to  this  difference  between  the  digestibility  of  the  cow's  casein  and  human 
casein,  as  the  first  seems  to  be  utilized  in  the  intestinal  tract  of  the  infant 
to  the  same  extent  as  human  casein  (P.  MtJLLER,  Rubner  and  Heubner  ^). 

*Compt.  rend.  Soc.  biolog.,  .^8. 

'See  Maly's  Jahresber.,  34. 

'  Ruppel,  Zeitschr.  f.  Biologie,  31;  Laves,  Zeitschr.  f.  physiol.  Chem.,  19. 

*See  Biedert,  Untersuchungen  iiber  die  chemischen  Untcrschiede  der  Menschen- 
und  Kuhmilch  (Stuttgart),  1884;  Langgaard,  Virchow's  Arch.,  fi.^;  Makris,  Studien 
iiber  die  Eiweisskorper  der  Frauen-  und  Kuhmilch,  Inaug.-Di.ss.  Strassburg,  1876. 

*  Miiller,  Zeitschr.  f.  Biologie,  .39;    Rubner  and  Heubner,  ibid.,  37. 


HUMAN    :\IILK.  531 

The  question  as  to  whether  the  above-mentioned  differences  depend  on 
a  decided  difference  in  the  two  caseins  or  only  on  an  unequal  relationship 
between  the  casein  and  the  salts  in  the  two  kinds  of  milk,  or  upon  other 
circumstances,  has  not  been  decided  as  yet.  According  to  Szoxtagh  and 
Zaitschek  and  also  Wroblewsky,  the  casein  from  human  milk  does  not 
yield  any  pseudonuclein  on  peptic  digestion,  and  hence  it  cannot  be  a 
nucleoalbumin,  Wroblewsky  has  found  the  following  for  the  composi- 
tion of  casern  from  woman's  milk:  C  52.24,  H  7.32,  X  14.97,  P  0.6S,  S  1.117 
per  cent.  According  to  Kobrak  ^  woman's  casein  yields  some  pseudonu- 
clein, and  with  repeated  solution  in  alkali  and  precipitation  by  an  acid  it 
becomes  more  and  more  like  cow's  casein.  He  therefore  suggests  the 
possibility  that  woman's  casein  is  a  compound  Ijetween  a  nucleoalbumin 
and  a  basic  protein. 

Woman's  milk  also  contains  lactalbumin,  tesides  the  casern,  and  a 
protein  substance,  verv^  rich  in  sulphur  (4.7  per  cent)  and  relatively  poor 
in  carbon,  which  Wroblewsky  calls  opalisin.  The  statements  as  to  the 
occurrence  of  proteoses  and  peptones  are  disputed  as  in  many  other  cases. 
Xo  positive  proof  as  to  the  occurrence  of  proteoses  and  peptones  in  fresh 
milk  has  been  given. 

Even  after  those  differences  are  eliminated  which  depend  on  the  imjjer- 
fect  analytical  methods  employed,  the  quantitative  composition  of  wonian's 
w'lk  is  variable  to  such  an  extent  that  it  is  impossible  to  give  any  average 
results.  The  recent  analyses,  especially  those  made  on  a  large  numter 
of  samples  by  Pfeiffer,  Adriaxce,  Camerer  and  Soldxer ,2  have  posi- 
tively sho^\'n  that  woman's  milk  is  essentially  poorer  in  proteins  but  richer 
in  sugar  than  cow's  milk.  The  quantity  of  protein  varies  tetween  10-20 
p.  m.,  often  amounting  to  only  15-17  p.  m.  or  less,  and  is  dependent  upon 
the  length  of  lactation  (see  below).  The  quantity  of  fat  also  varies  con- 
siderably, but  ordinarily  amounts  to  30-40  p.  m.  The  quantity  of  sugar 
should  not  be  below  50  p.  m.,  but  maj^  rise  to  even  80  p.  m.  About  60 
p.  m.  may  be  considered  as  an  average,  but  it  should  loe  borne  in  mind 
that  the  quantity  of  sugar  is  also  dependent  upon  the  length  of  lactation, 
as  it  increases  with  duration.  The  amount  of  mineral  bodies  varies  be- 
tween 2  and  4  p.  m. 

*  Szontagh,  Maly's  Jahresber.,  22;  Zaitschek,  1.  c;  Wrohle^vsky,  "Beitrage  zur 
Kenntnisdes  Frauenkaseins"  (Inaug.-Diss.  Bern,  1894),  and  "Ein  neiier  eiweis-fartiger 
Bestandteil  der  Milch,"  Anzeiger  der  Akad.  d.  "Wiss.  in  Krakau,  1898;  Kobrak, 
Pfliigcr's  Arch.,  SO. 

-Pfeiffer,  Jahrb.  f.  Kinderheilkunde,  20,  also  ^^aly's  Jahresber.,  13;  V.  Adriance 
and  J.  Adriance,  A  Clinical  Report  of  the  Chemical  Examination,  etc.,  Archives  of 
Pediatrics,  1897;  Camerer  and  Soldner,  Zeitschr.  f.  Biologic,  83  and  30.  In  regard 
to  the  composition  of  woman's  milk,  see  also  Biel,  Maly's  Jahresber.,  4;  Christenn, 
ibid..';  Mendesde  Leon,  iWt?.,  12;  Cerber,  Bull.  Soc.  chim.,  23;  Tolmatscheff,  Hoppe- 
Seyler's  Med.-chem.   Untersuch.,  272. 


532  MILK. 

From  a  quantitative  standpoint,  the  most  essential  differences  between 
woman's  and  cow's  milk  are  as  follows:  As  compared  with  the  quantity 
of  albumin,  the  quantity  of  casein  is  not  only  absolutely  but  also  relatively 
smaller  in  woman's  milk  than  in  cow's  milk,  while  the  latter  is  poorer  in 
milk-sugar.  Human  milk  is  richer  in  lecithin,  at  least  relatively  to  the 
amount  of  protein.  Burow  found  0.49-0.58  p.  m.  lecithin  in  cow's  milk 
and  0.58  p.  m.  in  woman's  milk,  which  corresponds  to  1.40  per  cent  for  the 
first  milk  and  3.05  per  cent  for  the  second,  calculated  on  the  percentage  of 
protein.  According  to  Koch  human  milk  and  cow's  milk  contain  lecithin 
as  well  as  cephalin.  The  total  quantity  of  both  bodies  in  human  milk  was 
0.78  p.  m.  and  in  cow's  milk  0.72-0.86  p.  m.  The  quantity  of  nucleon  is 
greater  in  woman's  milk.  According  to  Wittmaack  cow's  milk  contains 
0.566  p.  m.  nucleon,  and  woman's  milk  1.24  p.  m.,  and  according  to  Valenti 
the  quantity  of  nucleon  in  human  milk  is  indeed  still  higher.  Siegfried 
finds  that  the  nucleon  phosphorus  amounts  to  6.0  per  cent  of  the  total 
phosphorus  in  cow's  milk  and  41.5  per  cent  in  woman's  milk,  and  also 
that  in  human  milk  the  phosphorus  is  nearly  entirely  in  organic  combina- 
tion. Because  of  the  large  amount  of  casein  (and  calcium  phosphate) 
cow's  milk  is  much  richer  in  phosphorus  than  human  milk.  The  relation 
P205:N,  according  to  Schlossma.nn,i  is  equal  to  1:5.4  in  human  milk 
and  1 :2.7  in  cow's  milk.  Woman's  milk  is  poorer  in  mineral  bodies,  espe- 
cially lime,  and  it  contains  only  one-sixth  of  the  quantity  of  lime  as  com- 
pared with  cow's  milk.  The  mineral  constituents  of  human  milk  are 
better  assimilated  by  the  organism  of  the  nursing  child  than  those  of  cow's 
milk.  Human  milk  is  claimed  to  be  also  poorer  in  citric  acid  (Scheibe  ^), 
although  this  is  not  an  essential  difference. 

Another  difference  between  woman's  milk  and  other  varieties  of  milk  is 
Umikoff's  reaction,  which  seems  to  depend  upon  the  quantitative  composition, 
especially  the  relation  between  the  milk-sugar,  citric  acid,  lime,  and  iron  (Siebee  ^). 
This  reaction  consists  in  treating  5  c.c.  of  woman's  milk  ^vith-  2.5  c.c.  ammonia 
(10  per  cent)  and  heating  to  60°  C.  for  15-20  minutes,  when  the  mixture  becomes 
violet-red.     Cow's  milk  gives  a  yellowish-brown  color  when  thus  treated. 

According  to  Rubner  woman's  milk  contains  about  3  p.  m.  soaps,  but  this 
could  not  be  substantiated  by  Camerer  and  Soldner.  According  to  them 
woman's  milk  contains  no  soaps,  or  at  least  only  very  small  amounts.  They  also 
found  the 'quantity  of  urea  nitrogen  in  woman's  milk  to  be  0.11-0.12  p.  m., 
although  ScHONDORFF  ■•  found  nearly  twice  this  amount,  namely,  0.23  p.  m. 

In  regard  to  the  quantity  of  mineral  bodies  in  woman's  milk  we  have 

'  B  irow,  Zeitschr.  f.  physiol.  Chcm.,  30;  Koch,  ibid.,  47;  Wittmaack,  ibid.,  22; 
Siegfried,  ibid.,  22;  Valenti,  Biochem.  Centralbl.,  4;  Schlossmann,  Arch.  f.  Kinderheil- 
kunde,  40. 

^  Maly's  Jahresber.,  21. 

8  Zeitschr.  f.  physiol.  Chem.,  30. 

*  Rubner,  Zeitschr.  f.  Biologic,  30;  Camerer  and  Soldner,  i6vi.,  39;  Schondorff, 
Pfliiger's  Arch.,  81. 


HUMAN   MILK.  533 

the  analyses  of  several  investigators,  especially  of  Bunge  (analyses  A  and 
B)  and  of  Soldner  and  Camerer  (analysis  Cy.  Bunge  analyzed  the 
milk  of  a  woman,  fourteen  days  after  delivery,  whose  diet  contained  very 
little  common  salt  for  four  days  previous  to  the  analysis  {A),  and  again 
three  days  later  after  a  daily  addition  of  30  grams  of  NaCl  to  the  food  {B). 
The  figures  are  in  1000  parts  of  the  milk: 

A.                    B.  C. 

KjO 0.780  0.703  0.884 

Nap 0.232  0.257  0.357 

CaO 0.328  0.343  0.378 

MgO 0.064  0.065  0.053 

FePs 0.004  0.006  0.002 

PA 0.473  0.469  0.310 

CI 0.438  0.445  0.591 

The  relationship  of  the  two  bodies  potassium  and  sodium,  to  each  other 
may,  according  to  Bunge,  vary  considerably  (1.3-4.4  equivalents  of  potash 
to  1  of  soda).  By  the  addition  of  salt  to  the  food  the  quantity  of  sodium 
and  chlorine  in  the  milk  increases,  while  the  quantity  of  potassium  de- 
creases. De  Lange  found  more  Na  than  K  in  the  milk  at  the  beginning 
of  lactation.  Jolles  and  Friedjung  found  on  an  average  5.9  milligrams 
of  iron  per  liter  of  woman's  milk.  Camerer  and  Soldner  ^  find  about 
the  same  amount,  namely,  10-20  milligrams  Fe203  =  3.5-7  milligrams  iron 
in  1000  grams  human  milk. 

The  gases  of  woman's  milk  have  been  investigated  by  KiJLZ.^  He  found 
1.07-1.44  c.c.  of  oxygen,  2.35-2.87  c.c.  of  carbon  dioxide,  and  3.37-3.81 
c.c.  of  nitrogen  in  100  c.c.  of  milk. 

The  proper  treatment  of  cow's  milk  by  diluting  it  with  water  and  by 
certain  additions  in  order  to  render  it  a  proper  substitute  for  woman's  milk 
in  the  nourishment  of  children  cannot  be  determined  before  the  difference 
in  the  protein  bodies  of  these  two  kinds  of  milk  has  been  completely  studied. 

The  colostrum  has  a  higher  specific  gravity,  1.040-1.060,  a  greater 
quantity  of  coagulable  proteins,  and  a  deeper  j^ellow  color  than  ordinary 
woman's  milk.  Even  a  few  days  after  deliver}^  the  color  becomes  less 
yellow,  the  quantity  of  albumin  less,  and  the  number  of  colostrum-cor- 
puscles diminishes. 

We  have  the  older  analyses  of  Clemm  ^  and  the  recent  investigations  of 
Pfeiffer,  V.  and  J.  Adriance,  Camerer  and  Soldner  on  the  changes  in 
the  composition  of  milk  after  deliver}^     It  follows,  as  a  unanimous  result 


'  Bunge,  Zeitschr.  f.  Biologie,  10;  Camerer  and  Soldner,  ibid.,  39  and  44. 
'De  Lange,  Maly's  Jahresber.,  27;    Jolles  and  Friedjung,  Arch.  f.  exp.  Path,  u, 
Pharm.,  46:  Camerer  and  Soldner,  Zeitschr.  f .  Biologie,  46. 
^Zeitschr.  f.  Biologie,  32. 
"  See  Hoppe-Seyler    Physiol.  Chem.,  734. 


634  MILK. 

from  these  investigations,  that  the  quantity  of  protein,  which  amounts 
to  more  the  first  two  days,  sometimes  to  more  than  30  p.  m.  at  first,  rather 
quickl}^  and  then  more  gradually  diminishes  as  long  as  the  lactation  con- 
tinues, so  that  in  the  third  week  it  equals  about  10-18  p.  m.  Like  the 
protein  substances,  the  mineral  bodies  also  gradually  decrease.  The 
quantity  of  fat  shows  no  regular  or  constant  variation  during  lactation, 
while  the  lactose,  especially  according  to  the  observations  of  V.  and  J. 
Adrian'CE  (120  analyses),  increases  rather  quickly  the  first  days  and  then 
only  slowly  until  the  end  of  lactation.  The  analyses  of  Pfeiffer,  Camerer 
and  SoLDNER  also  show  an  increase  in  the  quantity  of  milk-sugar. 

The  two  mammary  glands  of  the  same  woman  may  yield  somewhat  different 
milk,  as  sho\\ai  by  Sourdat  and  later  by  Brunner.^  Likewise  the  different 
portions  of  milk  from  the  same  milking  may  have  varying  composition.  The 
first  portions  are  always  poorer  in  fat. 

According  to  l'Heritier  and  to  Vernois  and  Becquerel,  the  milk  of  blondes 
contains  less  casein  than  that  of  brunettes,  a  difference  which  Tolmatscheff  ^ 
could  not  substantiate.  Women  of  delicate  constitutions  yield  a  milk  richer  in- 
solids,  especially  in  casein,  than  women  with  strong  constitutions  (V.  and  B.). 

According  to  Vernois  and  Becquerel,  the  age  of  the  woman  has  an  effect  on 
the  composition  of  the  milk,  so  that  we  find  a  greater  quantity  of  proteins  and 
fat  in  women  15-20  years  old  and  a  smaller  quantity  of  sugar.  The  smallest 
quantity  of  proteins  and  the  greatest  quantity  of  sugar  are  found  at  20  or  from 
25  to  30  years  of  age.  Accorchng  to  Vernois  and  Becquerel,  the  milk  with  the 
first-born  is  richer  in  water — with  a  proportionate  diminution  of  casein,  sugar,  and 
fat — than  after  several  deliveries. 

The  influence  of  menstruation  seems  to  slightly  diminish  the  milk-sugar  and  to 
considerably  increase  the  fat  and  casein  (Vernois  and  Becquerel). 

Witch's  milk  is  the  secretion  of  the  mammary  glands  of  new-born  children  of 
both  sexes  immediately  after  birth.  This  secretion  has  from  a  ciualitative  stand- 
point the  same  constitution  as  milk,  but  may  show  important  differences  and 
variations  from  a  Cjuantitative  point  of  view.  Schlossberger  and  Hauff, 
GuBLER  and  Quevenne,  and  v.  Genser  ^  have  made  analyses  of  this  milk  and 
give  the  followmg  results:  10.5-28  p.  m.  proteins,  8.2-14.6  p.  m.  fat,  and  9-60 
p.  m.  sugar. 

As  milk  is  the  only  form  of  nourishment  during  a  certain  period  of  the 
life  of  man  and  mammals,  it  must  contain  all  the  nutriment  necessary  for 
life.  This  fact  is  shown  by  the  milk  containing  respresentatives  of  the 
three  chief  groups  of  organic  nutritive  substances — proteins,  carbohy- 
drates, and  fat;  and  all  milk  seems  to  contain  without  doubt  also  some 
lecithin  and  nucleon.  The  mineral  bodies  in  milk  must  also  occur  in  proper 
proportions,  and  on  this  point  the  experiments  of  Bunge  on   dogs  are  of 

'  Sourdat,  Compt.  rend.,  71;    Brunner,  Pfliiger's  Arch.,  7. 

^  l'Heritier,  cited  from  Hoppe-Seyk-r,  Physiol.  Chem.,  738;  Vernoi.s  and  Bec- 
querel, Du  lait  chez  la  femme  dans  I'etat  de  sante,  etc.  (Paris,  1853);  TolmatsehetT, 
Hoppe-Seyler,   Med  .-chem.    Untersuch.,   272. 

'Schlossberger  and  Hauff,  Annal.  d.  Chem.  u.  Pharm.,  9(5;  Gubler  and  Quevenne, 
cited  from  Hoppe-Seyler's  Physiol.  Chem.,  723;   v.  Gen.ser,  ibid. 


MILK  AND  FOOD.  535 

special  interest.  He  found  that  the  mineral  bodies  of  the  milk  occur  in 
about  the  same  relative  proportion  as  they  do  in  the  body  of  the  sucking 
animal.  Buxge  ^  found  in  1000  parts  of  the  ash  the  following  results 
{A  represents  results  from  the  new-boni  dog,  and  B  the  milk  from  the  bitch)  .v 

A.  B. 

KjO 114.2  149.8 

Nap 106.4  88.0 

CaO 295.2  272.4 

Ma;0 18.2         .  15.4 

Fe^O, 7.2  1.2 

P2O3 394.2  342.2 

CI 83.5  169.0 

Buxge  explains  the  fact  that  the  milk-ash  is  richer  in  potash  and  poorer 
in  soda  than  the  new-bom  animal  by  saying  that  in  the  growing  animal  the 
ash  of  the  muscles  rich  in  potash  relatively  increases  and  the  cartilage  rich 
in  soda  relatively  decreases.  In  regard  to  the  amount  of  iron  we  find  an 
unexpected  condition,  the  ash  of  the  new-born  animal  containing  six  times 
as  much  as  the  milk-ash.  This  condition  Bunge  explains  by  the  fact 
founded  on  his  and  Zalesky's  experiments,  that  the  quantity  of  iron  in  the 
entire  organism  is  highest  at  birth.  The  new-born  has  therefore  its  own 
supply  of  iron  for  the  growth  of  its  organs  even  at  birth. 

The  investigations  of  Hugounexq,  de  Laxge,  Camerer  and  Soldxer^ 
have  shown  that  in  man  the  conditions  are  different  from  those  in  animals, 
as  the  ash  of  the  child  has  an  entirely  different  composition  as  compared  to 
the  milk.  As  an  example  the  following  analyses  are  given  (of  Camerer  and 
Soldxer).  (A,  the  ash  of  the  sucking  infant,  and  B,  the  ash  of  the  milk.) 
The  results  are  in  1000  parts  of  the  ash. 

A.  B. 

K„0 78  314 

Na,0 91  119 

CaO 361  164 

MgO 9  26 

¥e,Oi 8  6 

PA 389  135 

CI 77  200 

We  cannot  therefore  state  as  a  definite  fact  that  the  composition  of  the 
ash  of  the  sucking  young  and  the  ash  of  the  corresponding  milk  coincide. 
Bunge  ^  nevertheless  claims  that  the  composition  of  the  ash  of  the  sucking 
young  of  various  mammals  is  nearly  the  same,  but  that  the  ash  of  the  milk 
differs  from  the  ash  of  the  young  in  so  far  as  the  slower  the  young  grows 
the  richer  it  is  in  alkali  chlorides  and  relativel}'  poorer  in  phosphates  and 

'  Zeitschr.  f.  physiol.  Chem.,  13. 

^  Hugounenq,  Compt.  rend.,  128;  de  Lange,  Zeitschr.  f.  Biologic,  40;  Camerer 
and  Soldner,  ibid.,  39,  40,  and  44. 

^  Bunge,  "Die  zunehmende  Unfahigkeit  der  Frauen  ihre  Kinder  zu  stillen,"  Miin- 
chen,  1900,  cited  by  Camerer,  Zeitschr.  f.  Biolo<rip,  40. 


536  MILK. 

lime-salts.  The  constituents  of  the  ash  have  two  functions  to  perform, 
namely,  the  building  up  of  the  tissues  and  secondly  the  preparation  of  the 
excreta,  especially  the  urine.  The  faster  the  young  grows  the  more  is  the 
first  in  evidence,  while  the  slower  it  develops,  the  second  is  prominent. 

The  quantity  of  mineral  bodies  in  the  milk,  and  especially  the  amount 
of  lime  and  phosphoric  acid,  as  shown  by  Bunge  and  Proscher  and  Pages, 
stands  in  close  relationship  to  the  rapidity  of  growth,  because  the  amount 
of  these  mineral  constituents  in  the  milk  is  greater  in  animals  which  grow 
and  develop  quickly  than  in  those  which  grow  only  slowly.  A  similar 
relationship  exists  also,  as  shown  by  the  researches  of  Proscher,  and  espe- 
cially of  Abderhalden,'  between  the  quantity  of  protein  in  the  milk  and 
the  rapidity  of  development  of  the  sucking  young.  The  amount  of  protein 
is  greater  in  the  milk  the  quicker  the  animal  develops. 

The  influence  of  the  food  on  the  composition  of  the  milk  is  of  interest 
from  many  points  of  view  and  has  been  the  subject  of  many  investigations. 
From  these  we  learn  that  in  human  beings  as  well  as  in  animals  an  insuffi- 
cient diet  decreases  the  quantity  of  milk  and  the  quantity  of  solids,  while 
abundant  food  increases  both.  From  the  observations  of  Decaisne  2  on 
nursing  women  during  the  siege  of  Paris  in  1871,  the  amount  of  casein,  fat, 
sugar,  and  salts,  but  especially  the  fat,  was  found  to  decrease  with  insufficient 
food,  while  the  quantity  of  lactalbumin  was  found  to  be  somewhat  increased. 
Food  rich  in  proteins  increases  the  quantity  of  milk,  and  also  the  solids 
contained,  especially  the  fat,  according  to  most  statements.  The  quantity 
of  sugar  in  woman's  milk  is  found  by  certain  investigators  to  be  increased 
after  food  rich  in  proteins,  while  others  claim  it  is  diminished.  A  diet  rich 
in  fat  may,  as  the  researches  of  Soxhlet  and  many  others  ^  have  shown, 
cause  a  marked  increase  in  the  fat  of  the  milk  when  the  fat  partaken  is  in  a 
readily  digestible  and  assimilable  form.  The  presence  of  large  quantities  of 
carbohydrates  in  the  food  seems  to  cause  no  constant,  direct  action  on  the 
quantity  of  the  milk  constituents .^  In  carnivora,  as  shown  by  Ssubotin,^ 
the  secretion  of  milk-sugar  proceeds  uninterruptedly  on  a  diet   consisting 


1  Proscher,  Zeitschr.  f.  physiol.  Chem.,  24;  Abderhalden,  ibid.,  27;  Pages,  Arch, 
de  Physi  )1.  (5),  7. 

=>  Cited  from  Hoppe-Seyler,  1.  c,  739. 

^  See  Maly's  Jahresber.,  2G.    See  also  Rasch,  Ergebnisse  der  Physiologie,  2,  Abt.  1. 

^In  regard  to  the  literature  on  the  action  of  various  foods  on  woman's  milk,  see 
Zalesky,  "Ueber  die  Einwirkung  der  Nahrung  auf  die  Zusammcnsetzung  und  Nahr- 
haftigkeit  der  Frauenmilch,"  Berlin,  klin.  Wochenschr. ,  188S,  which  also  contains  the 
literature  on  the  importance  of  diet  on  the  composition  of  other  kinds  of  milk.  In 
regard  to  the  rxtensive  literature  on  the  influence  of  various  foods  on  the  milk  pro- 
duction of  animals,  .see  Konig,  Chem.  d.  menschl.  Nahrungs  und  GenussmitteJ,  3.  Aufl., 
1,  298.  See  also  Maly's  Jahresber.,  29,  30,  31,  and  Morgen,  Beger  and  Fingerlmg, 
Landw.  Versuchsl.,  01. 

6  Centralbl.  f.  d.  med.  Wissensch.,  1866,  337. 


CHEMISTRY  OF    MILK  SECRETION.  537 

exclusively  of  lean  meat.  Watery  food  gives  a  milk  containing  an  excess 
of  water  and  having  little  value.  In  the  milk  from  cows  which  were  fed  on 
distillers'  grain  Commaille  ^  found  906.5  p.  m.  water,  26.4  p.  m.  casein,  4.3 
p.  m.  albumin,  18.2  p.  m.  fat,  and  33.8  p.  m.  sugar.  Such  milk  has  some- 
times a  peculiar  sharp  after-taste,  although  not  always.^ 

Chemistry  of  Milk-secretion.  That  the  constituents  which  occur  actu- 
ally dissolved  in  milk  pass  into  the  secretion  not  alone  by  filtration  or  diffu- 
sion, but  more  likely  are  secreted  by  a  specific  secretory  activity  of  the 
glandular  elements,  is  shown  by  the  fact  that  milk-sugar,  which  is  not 
found  in  the  blood,  is  to  all  appearances  formed  in  the  glands  themselves. 
A  further  proof  lies  in  the  fact  that  the  lactalbumin  is  not  identical  \^  ith 
seralbumin;  and  lastly,  as  Bunge  ^  has  shown,  the  mineral  bodies  secreted 
by  the  milk  are  in  quite  different  proportions  from  those  in  the  blood- 
serum. 

Little  is  kno-wn  in  regard  to  the  formation  and  secretion  of  the  specific 
constituents  of  milk.  The  older  theory,  that  the  casein  was  produced  from 
the  lactalbumin  by  the  action  of  an  enzyme,  is  incorrect  and  originated 
probably  from  mistaking  an  alkali  albuminate  for  casein.  Better  founded 
is  the  statement  that  the  casein  originates  from  the  protoplasm  of  the 
gland-cells.  There  does  not  seem  to  be  any  doubt  that  the  protoplasm  of 
the  cells  takes  part  in  the  secretion  in  such  a  manner  that  it  becomes  itself 
a  constituent  of  the  secretion,  and  this  also  agrees  with  Heidenhain's  * 
views.  According  to  Basch's  researches  the  casein  is  formed  in  the 
mammary  gland  by  the  nucleic  acid  of  the  nucleus  being  set  free  and 
uniting  intra-alveolar  with  the  transudated  serum,  thus  forming  a  nucleoalbu- 
min,  the  casein.  The  untenableness  of  this  view  has  been  shown  by  Lo- 
BiscH,  and  the  investigations  of  Hildebrandt  ^  upon  the  proteolytic 
enzyme  of  the  mammary  gland  and  the  autolysis  of  the  gland  have  not 
given  any  clue  as  to  the  mode  of  formation  of  casein. 

That  the  milk-fat  is  produced  by  a  formation  of  fat  in  the  protoplasm, 
and  that  the  fat -globules  are  set  free  by  their  destruction,  is  a  generally 
admitted  opinion,  which,  however,  does  not  exclude  the  possibility  that 
the  fat  is  in  part  taken  up  by  the  glands  from  the  blood  and  eliminated 
with  its  secretion.  That  the  fats  of  the  food  can  pass  into  the  milk  follows 
from  the  investigations  of  Winternitz,  as  he  has  been  able  to  detect  the 
passage  of  iodized  fats  in  the  milk.  Jantzen  has  showii  that  after  feeding 
iodized  casein,  the  milk-fat  of  goats  contained  a  little  iodine,  which  indicates 

1  Cited  from  Konig,  2,  235. 
'  See  Beck,  Maly's  Jahresber.,  25. 

'Lehrbnch  d.  physiol.  und  pathol.  Chem.,  3.  Aufl.,  93. 
^Hermarm^s  Handbuch,  5,  TeU  1,  380. 

^Basch,  Jahrb.  f.  Kinderheilkunde,  1-898;  Hildebrandt  Hofmeister's  Beitrage,  5; 
Lobisch,  ibid.,  8. 


538  MILK. 

that  the  iodized  milk-fat  could  also  have  a  different  origin.  As  a  con- 
tamination of  the  casein  fed  with  iodized  fat  was  not  excluded  in  these 
experiments,  they  do  not  seem  to  modify  the  proof  of  the  investigations  of 
WixTERXiTZ  and  others  (Caspaei,  Paraschtshuk  i).  The  abundant  quan- 
tities of  iodized  fat  which  were  eliminated  with  the  milk  in  these  cases  with- 
out doubt  depend,  at  least  in  great  part,  upon  the  iodized  fat  of  the  food, 
hence  it  cannot  be  said  that  all  of  the  milk-fat  containing  iodine  was 
unchanged  iodized  fat  of  the  food.  The  investigations  of  Spampani  and 
Daddi,  Paraschtschuk,  Gogitidse  and  others  on  the  passage  of  foreign 
fats  into  the  milk  also  indicate  the  passage  of  the  fat  of  the  food  into 
the  milk,  although  we  are  still  uncertain  on  this  point.  According  to 
SoxHLET  the  fat  of  the  food  does  not  pass  into  the  milk  directly,  but  is 
destroyed  in  place  of  the  body-fat,  which  then  becomes  available  and 
is,  as  it  were,  pushed  into  the  milk.  Hexriques  and  Haxsex  could  not 
detect  any  mentionable  quantity  of  linseed-oil  in  the  milk  after  feeding 
with  this  oil;  the  milk-fat  was  not  normal,  but  had  a  higher  iodine  equiva- 
lent and  a  higher  melting-point,  from  which  they  also  concluded  that  a 
transformation  of  the  food-fat  in  the  glandular  cells  is  possible.  The 
experiments  of  Gogitidse  2  with  soaps  also  speak  for  the  fact  that  the 
mammary  glands  have  the  property  of  forming  fats  by  S}nithesis  from  their 
components.  As  a  formation  of  fat  from  carbohydrates  in  the  animal 
organism  is  at  the  present  day  considered  as  positively  proved,  it  is  likewise 
possible  that  the  milk-glands  also  produce  fats  from  the  carbohydrates 
brought  to  them  by  the  blood.  It  is  a  well-known  fact  that  an  animal 
gives  off  for  a  long  time,  daily,  considerably  more  fat  in  the  milk  than  it 
receives  as  food,  and  this  proves  that  at  least  a  part  of  the  fat  secreted  by 
the  milk  is  produced  from  proteins  or  carbohydrates,  or  perhaps  from  both. 
The  question  as  to  how  far  this  fat  is  produced  directly  in  the  milk-glands, 
or  from  other  organs  and  tissues,  and  brought  to  the  gland  by  means  of 
the  blood,  cannot  be  decided. 

The  origin  of  milk-sugar  is  not  known.  Miixxz  calls  attention  to  the 
fact  that  a  number  of  very  widely  diffused  bodies  in  the  vegetable  king- 
dom— vegetable  mucilage,  gums,  pectin  bodies — yield  galactose  as  a  pro- 
duct of  decomposition,  and  he  believes,  therefore,  that  milk-sugar  may 
be  formed  in  herbivora  by  a  synthesis  from  dextrose  and  galactose.  This 
origin  of  milk-sugar  does  not  apply  to  carnivora,  as  they  produce  milk- 
sugar  when  fed  on  food  consisting  entirely  of  lean  meat.     The  observa- 

'  Winternitz,  Zeitschr.  f.  physiol.  Chem.,  24;  Jantzen,  Centralbl.  f.  Physiol.,  15; 
Caspari,  Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Supplbd.  and  Zeitschr.  f.  Biologic,  46; 
Paraschtschuk,  Chcm.  Centralbl.,  1903,  1. 

'Spampani  and  Daddi,  Maly's  Jahresber.,  2(5;  Henriques  and  Hansen,  ibifl,  29; 
Gogitidse,  Zeitschr.  f.  Biologic,  45  and  46.  See  also  Basch  Ergebnisse  d.  Physiol.,  2, 
Abt.  1. 


CHEMISTRY  OF    MILK    SECRETION.  539 

tioiis  of  Bert  and  Thierfelder  ^  that  a  mother-substance  of  the  milk- 
sugar,  a  saccharogen,  occurs  in  the  glands  cannot  give  further  explanation  as 
to  the  formation  of  milk-sugar,  as  the  nature  of  this  mother-substance  is  still 
imkno'^ii.  As  the  animal  bod}'  has  undoubtedly  the  power  of  converting 
one  variety  of  sugar  into  another,  the  origin  of  the  milk-sugar  can  be 
sought  simply  m  the  dextrose  introduced  as  food  or  formed  in  the  bod}'. 
Certain  observations  indicate  such  an  origin,  among  others  those  of 
PoRCHER,2  ^.}^Q  fovmd  that  dextrose  appeared  in  the  urine  after  delivery 
when  the  mammary  glands  of  the  goat  had  previously  been  extirpated. 
This  glycosuria  is  explained  simply  by  the  fact  that  the  lactose-forming 
action  of  the  gland  was  removed  at  the  time  of  delivery,  when  large  amounts 
of  dextrose  were  produced. 

The  passage  of  foreign  substances  into  the  milk  stands  in  close  connec- 
tion with  the  chemical  processes  of  milk  secretion. 

It  is  a  well-kno-^-n  fact  that  milk  acquires  a  foreign  taste  from  the  food 
of  the  animal,  which  is  in  itself  a  proof  that  foreign  bodies  pass  into  the 
milk.  This  fact  becomes  of  special  importance  in  reference  to  such  injurious 
substances  as  may  be  introduced  mto  the  organism  of  the  nursing  child  by 
means  of  the  milk. 

Among  these  substances  may  be  mentioned  opium  and  moi-phine,  which 
after  large  doses  pass  into  the  milk  and  act  on  the  child.  Alcohol  may  also 
pass  into  the  milk,  but  probably  not  in  such  c^uantities  as  to  have  any  direct 
action  on  the  nursing  child. '^  Alcohol  is  claimed  to  have  been  detected  in 
the  milk  after  feeding  cows  with  brewer's  grains. 

Among  inorganic  bodies,  iodine,  arsenic,  bismuth. antimony,  zinc,  lead, 
mercur}^,  and  iron  have  been  found  in  milk.  In  icterus  neither  bile-acids 
nor  bile-pigments  pass  into  the  milk. 

Under  diseased  conditions  no  constant  change  has  been  found  in  woman's 
milk.  In  isolated  cases  Schlossberger,  Joly  and  Filhol  *  have  observed 
indeed  a  markedl}'  abnormal  composition,  but  no  positive  conclusion  can  be 
derived  therefrom. 

The  changes  in  cow's  milk  in  disease  have  been  little  studied.  In  tuberculosis 
of  the  udder  Storch  ^  found  tubercle  bacilli  in  the  milk,  and  he  also  noted  that 
the  milk  became  more  and  more  diluted,  during  the  disease,  with  a  serous  licjuid 
similar  to  blood-serum,  so  that  the  glands  finally,  instead  of  yielding  milk,  gave 
only  blood-serum  or  a  serous  fluid.     Hussox  ^  found  that  milk    from  murrain 

'  Miintz,  Compt.  rend.,  102;    Bert  and  Thierfelder,  foot-note  3,  p.  514. 

^Compt.  rend.,  138  and  141. 

^  See  Klingemann,  Virchow's  Arch.,  126,  and  Rosemann,  Pfliiger's  Arch.,  78. 

^Schlossberger,  Annal.  d.  Chem.  u.  Phami.,  90;  Joly  and  Filhol,  cited  from 
v.  Gonip-Besanez,  Lehrb.,  4.  Aufl.,  438. 

^  See  Bans;,  Om  Tuberkulose  i  Kopns  Yver  og  om  tuberkulos  Malk.  Xord.  med. 
Arkiv,  10,  and  also  Maly's  Jahresber.,  14, 170;  Storch  Maly's  Jahresber.,  14. 

^  Compt.  rend.,  7.3. 


540  MILK. 

cows  contained  more  proteins  but  considerably  less  fat  and  (in  severe  cases)  less 
sugar  than  norma]  milk. 

The  milk  may  be  blue  or  red  in  color,  due  to  the  development  of  micro- 
organisms. 

The  formation  of  concrements  in  the  exit-passages  of  the  cow's  udder  is  often 
observed.  These  consist  chiefly  of  calcium  carbonate,  or  of  carbonate  and  phos- 
phate with  only  a  small  amount  of  organic  substances. 


CHAPTER  XV. 

URINE. 

Urine  is  the  most  important  excretion  of  the  animal  organism;  it  is  the 
means  of  eUminating  the  nitrogenous  metaboUc  products,  also  the  water  and 
the  soluble  mineral  substances;  and  in  many  cases  it  furnishes  important 
data  relative  to  the  metabolism,  quantitatively  by  its  variation,  and  quali- 
tatively by  the  appearance  of  foreign  bodies  in  the  excretion.  IMoreover  in 
many  cases  we  are  able  from  the  chemical  or  morphological  constituents 
which  the  urine  abstracts  from  the  kidneys,  ureter,  bladder,  and  urethra 
to  judge  of  the  condition  of  these  organs;  and  lastly,  urinary  analysis 
affords  an  excellent  means  of  deciding  the  question  as  to  how  certain 
medicinal  agents  or  other  foreign  substances  introduced  into  the  organism 
are  absorbed  and  chemically  changed.  In  this  respect  especially  urinary 
analysis  has  furnished  very  important  particulars  in  regard  to  the  nature  of 
the  chemical  processes  taking  place  within  the  organism,  and  it  is  therefore 
not  only  an  important  aid  in  diagnosis  to  the  physician,  but  it  is  also  of 
the  greatest  importance  to  the  toxicologist  and  the  physiological  chemist. 

In  studying  the  secretions  and  excretions  the  relationship  must  be  sought 
between  the  chemical  structure  of  the  secreting  organ  and  the  chemical 
composition  of  its  secreted  products.  Investigations  with  respect  to  the 
kidneys  and  the  urine  have  led  to  very  few  results  from  this  standpoint. 
Although  the  anatomical  relation  of  the  kidneys  has  been  carefully  studied, 
their  chemical  composition  has  not  been  the  subject  of  thorough  analytical 
research.  In  cases  in  which  a  chemical  investigation  of  the  kidneys  has 
been  undertaken,  it  has  been  in  general  only  of  the  organ  as  such,  and  not 
of  the  different  anatomical  parts.  An  enumeration  of  the  chemical  con- 
stituents of  the  kidneys  known  at  the  present  time  can,  therefore,  have  only 
a  secondary  value. 

In  the  kidneys  we  find  proteins  of  different  kinds.  According  to 
Halliburton  the  kidneys  do  not  contain  any  albumin,  but  only  a  globulin 
and  a  nucleoproteid.  The  globulin  coagulates  at  about  52°  C,  and  the 
nucleoproteid  contains  0.37  per  cent  jDhosphorus.  According  to  L.  Leiber- 
MANN  the  kidneys  contain  a  lecithalbumin,  and  he  ascribes  to  this  bod}^  a 
special  importance  in  the  secretion  of  acid  urines.     The  kidneys  also  contain 

541 


542  URINE. 

according  to  Lonnberg,  a  mucin-like  substance.  This  substance  yields  no 
reducing  body  on  boiling  with  acids  and  belongs  chiefly  to  the  papillae,  and 
is,  according  to  Loxnberg,  a  nucleoalbumin  (nucleoproteid?).  The  cor- 
tical substance  is  richer  in  another  nucleoalbumin  (nucleoproteid)  unUke 
mucin.  It  has  not  been  decided  what  relationship  this  last  substance 
bears  to  Halliburton's  nucleoproteid.  The  nucleic  acid  obtained  by 
Mandel  and  Levene  from  beef  kidneys  yielded  guanine,  adenine,  thy- 
mine, and  cytosine  on  cleavage.  According  to  Morner  ^  chondroitin- 
sulphuric  acid  occurs  as  traces.  jMandel  and  Levene  ^  have  also  obtained 
glucothionic  acid  from  the  kidneys.  Fat  occurs  only  in  very  small  amounts 
in  the  cells  of  the  tortuous  urinary  passages.  Among  the  extractive  bodies 
of  the  kidneys  one  finds  imrine  bases,  also  urea,  uric  acid  (traces),  glycogen, 
leucine,  inosite,  taurine,  and  cystine  (in  ox-kidneys).  The  quantitative 
analyses  of  the  kidneys  thus  far  made  possess  little  interest.  Oidtmann  ^ 
found  810.94  p.  m.  water,  179.16  p.  m.  organic  and  0.99  p.  m.  inorganic 
substance  in  the  kidney  of  an  old  woman. 

The  fluid  collected  under  pathological  conditions,  as  in  hydronephrosis,  is  thin 
with  a  variable  but  generally  low  specific  gravity.  Usually  it  is  straw-yellow  or 
paler  in  color,  and  sometimes  colorless.  Most  frequently  it  is  clear,  or  only 
faintly  cloudy  from  white  blood-corpuscles  and  epithelium-cells;  in  a  few  cases 
it  is  so  rich  in  form-elements  that  it  appears  like  pus.  Protein  occurs  gener- 
ally in  small  amounts;  occasionally  it  is  entirely  absent,  but  in  a  few  rare  cases 
the  amount  is  nearly  as  large  as  in  the  blood-serum.  Urea  occurs  sometimes 
in  considerable  amounts  when  the  parenchyma  of  the  kidneys  is  only  in  part 
atrophied ;  in  complete  atrophy  the  urea  may  be  entirely  absent. 

I.    Physical  Properties  of  Urine. 

Consistency,  Transparency,  Odor,  and  Taste  of  Urine.  Under  physio- 
logical conditions  urine  is  a  thin  liquid  and  gives,  when  shaken  with  air,  a 
froth  which  quickly  subsides.  Human  urine,  or  urine  from  carnivora,  which 
is  habitually  acid,  appears  clear  and  transparent,  often  faintly  fluorescent, 
immediately  after  voiding.  When  allowed  to  stand  for  a  little  while  human 
urine  shows  a  light  cloud  (nubecula),  which  consists  of  the  so-called  "  mucus," 
and  generally  also  contains  a  few  epithelium  cells,  mucus-corpuscles,  and 
urate-granules.  The  presence  of  a  larger  quantity  of  urates  renders  the 
urine  cloudy,  and  a  clay-yellow,  yellowish-brown,  rose-colored,  or  often 
brick-red  precipitate  (sedimentum  lateritium)  settles  on  cooling,  because  of 
the  greater  insolubility  of  the  urates  at  the  ordinary  temperature  than  at 
the  temperature  of  the  body.     This  cloudiness  disappears  on  gently  warm- 

1  Halliliurton,  Journ.  of  Physiol.,  13,  Suppl.,  and  18;  Liebermann,  Pfliiger's  Arch. 
50  and  54;  Lonnberg,  see  Maly's  Jahresber.,  20;  Mandel  and  Levene,  Zeitschr.  f. 
phy.siol.  Cheni.,  47;   Morner,  Skand.  Arch.  f.  Physiol.,  6. 

^Zeitschr.  f.  physiol.  Chem.,  45. 

^  Cited  from  v.  Gorup-Besanez,  Lehrbuch,  4.  Aufi.,  732. 


REACTION.  543 

ing.  In  new-born  infants  the  cloudiness  of  the  urine  during  the  first  4-5 
days  is  due  to  epithelium,  mucus-corpuscles,  uric  acid,  and  urates.  The 
urine  of  herbivora,  which  is  habitually  neutral  or  alkaline  in  reaction,  is 
very  cloudy  on  account  of  the  carbonates  of  the  alkaline  earths  present. 
Human  urine  may  sometimes  be  alkaline  under  physiological  conditions. 
In  this  case  it  is  cloudy,  due  to  the  earthy  phosphates,  and  this  cloudiness 
does  not  disappear  on  warming,  differing  in  this  respect  from  the  sedimen- 
tum  lateritium.  Urine  has  a  salt}"  and  faintly  bitter  taste  produced  by 
sodium  chloride  and  urea.  The  odor  of  urine  is  peculiarly  aromatic;  the 
bodies  which  produce  this  odor  are  unkno^^^l. 

The  color  of  urine  is  normally  pale  yellow  when  the  specific  gravity  is 
1.020.  The  color  otherwise  depends  on  the  concentration  of  the  urine  and 
varies  from  pale  straw-yellow,  when  the  urine  contains  small  amounts  of 
solids,  to  a  dark  reddish  yellow  or  reddish  bro\\Ti  in  stronger  concentration. 
As  a  rule  the  intensity  of  the  color  corresponds  to  the  concentration,  but 
under  pathological  conditions  exceptions  occur  such  as  is  found  in  diabetic 
urine,  which  contains  a  large  amount  of  solids  and  has  a  high  specific 
gravity  and  a  pale-yellow  color. 

The  reaction  of  urine  depends  essentially  upon  the  composition  of  the 
food.  The  carnivora,  as  a  rule,  void  an  acid,  the  herbivora,  a  neutral  or 
alkaline  urine.  If  a  carnivore  is  put  upon  a  vegetable  diet,  its  urine  mav 
become  less  acid  or  neutral,  while  the  reverse  occurs  when  an  herbivore  is 
starved,  that  is,  when  it  lives  upon  its  own  flesh,  as  then  the  urine  voided  is 
acid. 

The  urine  of  a  healthy  man  on  a  mixed  diet  has  an  acid  reaction,  and 
the  sum  of  the  acid  equivalents  is  greater  than  the  sum  of  the  basic  equiva- 
lents. This  depends  upon  the  fact  that  in  the  physiological  combustion  of 
neutral  substances  (proteins  and  others)  within  the  organism,  acids  are  pro- 
duced, chiefly  sulphuric  acid,  but  also  phosphoric  and  organic  acids,  such  as 
hippuric,  uric,  and  oxalic  acids,  aromatic  oxyacids,  and  others.  From  this  it 
follows  that  the  acid  reaction  is  not  due  to  one  acid  alone.  The  ordinary 
view  that  the  acid  reaction  is  due  chiefly  to  dihydrogen  phosphates  is 
therefore  not  true.  The  various  acids  take  part  in  the  acid  reaction  in 
proportion  to  their  dissociation,  since,  according  to  the  ion  theory,  the  acid 
reaction  of  a  mixture  is  dependent  upon  the  number  of  hydrogen  ions 
present. 

The  composition  of  the  food  is  not  the  only  influence  which  affects  the 
degree  of  acidity  of  human  urine.  For  example,  after  taking  food,  at  the 
beginning  of  digestion,  when  a  larger  amount  of  gastric  iuice  containino- 
hydrochloric  acid  is  secreted,  the  urine  may  be  neutral  or  even  alkaline .^ 
The  statements  of  various  investigators  are  rather  contradictor^^  in  re2;ard 

1  Contradictory  statements  are  found  in  Linossier,  Maly's  Jahresber.,  27. 


544  URINE. 

to  the  time  of  the  appearance  of  the  maximum  and  minimum  of  the  acid- 
ity, which  ma}'  in  part  be  explained  by  the  varying  individuahty  and 
conditions  of  Ufe  of  the  persons  investigated.  It  has  not  infrequ2ntly  been 
observed  that  perfectly  healthy  persons  in  the  morning  void  a  neutral  or 
alkaline  urine  which  is  cloudy  from  earthy  phosphates.  The  effect  of 
muscular  activity  on  the  acidity  of  urme  has  not  been  positively  determined. 
According  to  Hoffmann,  Ringstedt,  Oddi  and  Tarulli,  and  Vozarik 
muscular  work  raises  the  degree  of  acidity,  but  Aoucco  ^  claims  that  it 
decreases  it.     Abundant  perspiration  reduces  the  acidity  (Hoffivl\nn) . 

In  man  and  especially  in  carnivora  it  seems  that  the  degree  of  acidity  of 
the  urine  cannot  be  increased  above  a  certain  point,  even  though  mineral 
acids  or  organic  acids  wliich  are  burnt  up  with  difficulty  are  ingested  in  large 
quantities.  When  the  supply  of  carbonates  of  the  fixed  alkalies  stored  up 
in  the  organism  for  this  purpose  is  not  sufficient  to  combine  with  the  excess 
of  acid,  then  ammonia  is  spht  off  from  the  proteins  or  their  decomposition 
products,  and  this  excess  of  acid  combines  therewith,  forming  ammonium 
salts,  which  pass  into  the  urine.  In  herbivora  such  a  combination  of  the 
excess  of  acid  with  ammonia  does  not  seem  to  take  place,  or  not  to  the  same 
extent,  and  therefore  herbivora  soon  die  when  acids  are  given.  This  is 
true  at  least  for  rabbits,  while  according  to  Baer  -  this  power  of  increasing 
the  elimination  of  ammonia  exists  also  in  the  goat,  monkey,  and  pig,  hence 
no  definite  difference  in  this  regard  exists  between  herbivora  and  carnivora. 
Nevertheless  the  degree  of  acidity  of  human  urine  may  be  easily  diminished 
so  that  the  reaction  becomes  neutral  or  alkaline.  This  occurs  after  the 
taking  of  carbonates  of  the  fixed  alkalies  or  of  such  alkaU  salts  of  vegetable 
acids — tartaric  acid,  citric  acid,  and  malic  acid — as  are  easily  burnt  into 
carbonates  in  the  organism.  Under  pathological  conditions,  as  in  the 
absorption  of  alkaline  transudates,  or  the  alkaline  fermentation  within  the 
bladder,  the  urine  may  become  alkaline. 

A  urine  with  an  alkaUne  reaction  caused  by  fixed  alkalies  has  a  very 
different  diagnostic  value  from  one  whose  alkaline  reaction  is  caused  by 
the  presence  of  ammonium  carbonate.  In  the  latter  case  we  have  to  deal 
with  a  decomposition  of  the  urea  of  the  urine  by  the  action  of  micro-organ- 
isms. 

If  one  wishes  to  determine  whether  the  alkaline  reaction  of  the  urine  is 
due  to  ammonia  or  to  fixed  alkalies,  a  piece  of  red  litmus  paper  is  dipped  into 
the  urine  and  allowed  to  dry  exposed  to  the  air  or  to  a  g-^^ntle  heat.  If  the 
alkaline  reaction  is  due  to  ammonia,  the  paper  becomes  red  again;  but  if 
it  is  caused  by  fixed  alkalies,  it  remains  l)lue. 

'  Hoffmann,  see  Maly's  Jahresber.,  14;  Ringstedt,  ibid.,  20;  Oddi  and  Tarulli, 
ihid.,  24;   Aducco,  ibid.,  17;   Vozarik,  Pfliiger's  Arch.,  111. 

^  See  Winterberg,  Zeitsehr.  f.  physiol.  Chem.,  2.5.  and  T.  Baer,  Arch.  f.  exp.  Path.  u. 
Pharm.,  h\. 


DETERMINATION   OF  THE  ACIDITY.  545 

Determination  of  the  Acidity.  As  the  quantity  of  phosphoric  acid 
present  as  dihydrogen  salt,  as  above  stated,  cannot  be  used  as  a  measure 
of  the  acidity,  all  the  older  methods  suggested  for  the  estimation  of  his 
portion  of  the  phosphoric  acid  are  not  suited  for  acidity  determinations. 
We  now  determine  the  acidity  simply  by  acidimetric  methods,  titrating 
with  N/10  caustic  alkali,  using  phenolphthalein  as  an  indicator  (Naegeli, 
HoBER.  FoLix).  On  account  of  the  color  of  the  urine  and  the  presence  of 
ammonium  salts  and  alkaline  earths,  this  method  cannot  yield  entirely 
exact  results.  The  greatest  error  depends  upon  the  alkaline  earths,  which, 
on  titration  with  caustic  alkali,  precipitate  as  earthy  phosphates  in  variable 
amounts  and  of  variable  composition.  This  error  can  be  prevented,  ac- 
cording to  FoLiN,  by  the  addition  of  neutral  potassium  oxalate,  which 
precipitates  the  lime,  and  in  this  way  the  disturbing  action  of  the  ammo- 
nium salts  is  also  inliiljited.  Perfectly  acccurate  results  are  not  obtained 
by  tliis  method,  but  it  is  the  best  of  those  which  have  been  suggested. 

It  is  performed  as  follows:  25  c.c.  of  urine  are  placed  in  an  Erlenmeyer 
flask  (about  200  c.c.  capacity),  treated  with  1-2  drops  of  h  percent  phen- 
olphthalein solution,  and  shaken  with  15-20  grams  of  powdered  potassium 
oxalate  and  immediately  titrated  with  N/10  caustic  soda  with  constant 
shaking  until  a  pronounced  pale-rose  color  appears.  VozXrik  ^  titrates  the 
diluted  urine  without  the  addition  of  oxalate  and  uses  phenolphthalein  as 
indicator. 

The  acidity,  as  determined  by  titration,  varies  considerably  under 
physiological  conditions,  but  calculated  as  hydrochloric  acid  it  amounts 
to  about  1.5-2.3  grams  in  man  in  the  24  hours. 

By  titration  we  learn  the  amount, of  hydrogen  present  which  can  be 
substituted  by  a  metal,  i.e.,  the  acidity  in  the  ordinaiy  older  sense,  but  not 
the  true  acidity,  the  ion  acidity,  which  is  given  by  the  concentration  of  the 
hydrogen  ions  of  the  urine.  For  similar  reasons,  as  indicated  pre\-iouslv  in 
treating  of  the  alkalinity  of  the  blood-serum  (page  191),  the  ion  acidity 
cannot  be  determined  by  titration,  wliile  it  can  be  determined  accordino- 
to  the  principle  of  the  electrometric  gas-chain  method  as  there  given.  Such 
estimations  have  been  made  by  v.  Rhorer  and  by  Hober.^  For  normal 
urine  v.  Rhorer  found  as  a  minimum  4x10"*^,  as  a  maximum  76x10"^ 
and  as  an  average  30x10"''.  Hober  found  4.7X10"^,  100  X"^,  and 
49X10"'',  respectively.  On  an  average  the  urine  contains  therefore  30-50 
grams  of  hydrogen  ions  in  10  million  liters,  and  as  in  the  same  quantity 
of  purest  water  there  is  contained  in  round  numl:)ei-s  1  gram  of  hvdrogen 
ions,  the  urine  contains,  therefore.  30-50  times  as  many  hydrojren  ions  as 


^Naegeli,  Zeitschr.  f.  physiol.  Chem.,  30;   Hober,  Hofmeister's  Beitrage,  3-  Folin 
Amer.  Journ.  of  Physiol.,  9;   Vozarik,  1.  c. 

^  V.  Rhorer,  Pfliiger's  Arch.,  86;    Hober,  1.  c. 


546  URINE. 

the  water.  From  Hober's  investigations  it  also  follows  that  no  direct 
relationship  exists  between  the  titration  acidity  and  the  ion  acidity,  and 
that  the  extent  of  these  two  acidities  may  be  independent  of  each  other. 

The  osmotic  pressure  of  the  urine  varies  considerably  even  under 
physiological  conditions.  The  limits  for  the  freezing-point  depression  has 
been  found  by  a  number  of  investigators  to  be  J  =  0.87°-  2.71°  C.^  After 
partaking  of  considerable  water  it  may  be  markedly  lower;  and  on  diminished 
supply  of  water  it  may  be  consideralily  higher. 

According  to  Bugarsky  a  certain  relationship  exists  between  the  freezing- 

J 
point   depression   and   the   specific   gravity,   namely,  -=  constant  =  75.     This 

equation,  where  s  represents  the  specific  gravit}^,  has  no  general  application,  and 
according  to  Steyrer  ^  is  only  approximate  for  normal  urines.  The  validity 
of  the  relationship  found  by  Bugarsky  between  the  electrical  conductivity 
and  the  ash  content  of  the  urine,  seems  also  to  require  further  proof. 

The  specific  gravity  of  urine,  which  is  dependent  upon  the  relationsliip 
existing  between  the  quantity  of  water  secreted  and  the  solid  urinary  con- 
stituents, especially  the  urea  and  sodium  chloride,  may  vary  considerably, 
but  is  generally  1.017-1020.  After  drinking  large  quantities  of  water  it 
may  fall  to  1.002,  while  after  profuse  perspiration  or  after  drinking  very 
little  water  it  may  rise  to  1.0S5-1.040.  In  new-l^orn  infants  the  snecific 
gravity  is  low,  1.007-1.005.  The  determination  of  the  specific  gravity  is 
an  important  means  of  learning  the  a^'erage  amount  of  solids  eliminated 
from  the  organism  in  the  urine,  and  on  this  account  the  determination 
becomes  of  true  value  only  when  at  the  same  time-  the  quantity  of  urine 
voided  in  a  given  time  is  determined.  The  different  portions  of  urine 
voided  in  the  course  of  the  twenty-four  hours  are  collected,  mixed  together, 
the  total  quantity  measured,  and  then  the  specific  gravity  taken. 

The  determination  of  the  specific  gravity  is  most  accurately  o]:)tained  with 
the  pycnometer.  For  ordinary  cases  the  specific  gravity  may  be  deter- 
mineci  with  sufficient  accuracy  by  means  of  areometers.  The  areometers 
found  in  the  trade,  or  n  riff  meter:--,  are  graduated  from  1.000  to  1.040;  for 
exact  ol)servations  it  is  better  to  use  two  urinometers,  one  graduated  from 
1.000  to  1.020,  and  the  other  from  1.020  to  1.040. 

To  determine  the  specific  gravity  of  urine,  if  necessary  filter  the  urine, 
or  if  it  contains  a  urate  sediment,  first  dissolve  it  by  gentle  heat,  then  pour 
the  clear  urine  into  a  dry  cylinder,  avoiding  the  formation  of  froth.  Air- 
bubbles  or  froth,  when  present,  must  be  remo^■ed  with  a  glass  rod  or  filter- 
paper.  The  cylinder,  which  should  he  about  four-fifths  fidl.  must  be  wide 
enough  to  allow  the  urinometer  to  swim  freely  in  the  liquid  without  touch- 
ing the  sides.  The  cylinder  and  urinometer  shoidd  both  be  drv  or  j^reviously 
washed  with  the  urine.     On  reading:,  the  eye  is  brought  on  a  level  with  the 

■See  Strauss,  Zeitschr.  f.  klin.  Med.,  47. 

^  Bugarsky.  Pfliijrer's  Arch.,  68;   Steyrer,  Hofmcister's  Beitrage,  2. 


ORGANIC   PHYSIOLOGICAL  CONSTITUENTS.  547 

lower  meniscus — which  occurs  when  the  surface  of  the  liquid  and  the  lower 
limb  of  the  meniscus  coincide;  the  readinj:;  is  then  made  from  the  point 
where  this  curved  line  coincides  wi;h  the  scale  ot  the  urinometer.  If  the 
eye  is  not  in  the  same  hoii/.ontal  plane  with  the  convex  line  of  the 
meniscus,  but  is  too  hi,s;h  or  too  low,  the  surface  of  the  lic[uid  assumes  the 
shape  of  an  ellipse,  and  the  reading  in  this  position  is  incorrect.  Before 
readino;,  press  the  urinometer  gently  down  into  the  liquid  and  then  allow 
it  to  rise,  and  wait  until  it  is  at  rest. 

Each  urinometer  is  graduated  for  a  certain  temperature,  which,  at  least 
in  the  case  of  the  better  ones,  is  marked  on  the  instrument.  If  the  urine 
is  not  at  the  proper  temperature,  the  following  corrections  must  be  made: 
For  ever}'  three  degrees  above  the  normal  temperature  one  unit  ot  the  last 
order  is  added  t')  the  reading,  and  for  ever\'  three  degrees  below  the  nor- 
mal temperature  one  unit  (as  above)  is  subtracted  from  the  specific  g^a^dty 
observed.  For  example,  when  a  urinometer  graduated  for  15°  C.  shows 
a  specific  2:ra\ntv  of  1.017  at  24°  C,  then  the  specific  gravitv  at  15"  C.= 
1.017  +  0.003=1.020. 

When  great  exactitude  is  required,  as,  for  instance,  a  determination  to 
the  fourth  decimal  point,  we  make  use  of  a  urinometer  constructed  by 
LoHxsTEiN.i  JoLLES  ^  has  also  devised  a  small  urinometer  for  the  deter- 
mination of  the  specific  gravity  of  small  amounts  of  urine,  20-25  c.c.  The 
specific  gravit}^  may  also  be  determined  by  the  West^^h.al  hydrostatic 
balance. 

n.  Organic  Physiological  Constituents  of  Urine. 

+  X'TT 

Urea,  Ur,  C0N2H4  =  C0<*^f;-,  has  been  synthetically  prepared  in  sev- 

iN  rl2 

eral  wa3'S,  especially,  as  Wohlee  showed  in  1828,  by  the  metameric  trans- 
formation of  ammonium  isocyanate:  CO.N.NH4--^CO(NH2)2-  It  is  also 
produced  by  the  decomposition  or  oxidation  of  certain  bodies  found  in  the 
animal  organism,  such  as  purine  bodies,  creatine,  agrinine,  other  amino- 
acids,  and  polypeptides. 

Urea  is  found  most  abundantly  in  the  urine  of  carnivora  and  man,  but  in 
smaller  quantities  in  that  of  herbivora.  The  quantity  in  human  urine  is 
ordinarily  20-30  p.  m.  It  has  also  been  found  in  small  quantities  in  the 
urine  of  amphibians,  fishes,  and  certain  birds.  Urea  occurs  in  the  perspira- 
tion in  small  quantities,  and  as  traces  in  the  blood  and  in  most  of  the  animal 
fluids.  It  also  occurs  in  rather  large  quantities  in  the  blood,  liver,  muscle.-^ 
and  bile  *  of  sharks.  Urea  is  also  found  in  certain  tissues  and  organs  of 
mammals,  especially  in  the  liver  and  spleen,  although  only  in  small  amounts. 
Under  pathological  conditions,  as  in  obstructed  excretion,  urea  may  appear 
to  a  considerable  extent  in  the  animal  fluids  and  tissues. 

'  Pfliigpr's  Arch  ,  ."iO;  Chem.  Centralbl.,  1895, 1,  and  1896,  2. 

MVien.  med.  Pressc,  1897,  No.  8. 

^  V.  Schrocder,  Zeitschr.  f.  physiol.  Chem.,  14. 

*  Hammarsten,  ihtd.,  24. 


548  URINE. 

The  quantity  of  urea  which  is  voided  in  twenty-four  hours  on  a  mixed 
diet  is  in  a  grown  man  about  30  grams,  in  women  somewhat  less.  While 
children  void  less,  the  excretion  relative  to  their  body  weight  is  greater  than 
in  grown  persons.  The  physiological  significance  of  urea  lies  in  the  fact 
that  this  body  forms  in  man  and  carnivora,  from  a  quantitative  standpoint, 
the  most  important  nitrogenous  end-product  of  the  metabolism  of  protein 
bodies.  On  this  account  the  slimination  of  urea  varies  to  a  great  extent 
with  the  catabolism  of  the  protein,  and  above  all  with  the  quantity  of 
absorbable  proteins  in  the  food  ingested.  The  elimination  of  urea  is  great- 
eat  after  an  exclusive  meat  diet,  and  lowest,  indeed  less  than  during  starva- 
tion, after  the  consumption  of  non-nitrogenous  substances,  since  these 
diminish  the  metaljolism  of  the  proteins  of  the  body. 

If  the  consumption  of  the  proteins  of  the  body  is  increased,  then  the 
elimination  of  nitrogen  is  correspondingly  increased.  This  is  found  to  be 
the  case  in  fevers,  after  poisoning  with  arsenic,  antimony,  phosphorus,  and 
other  protoplasmic  poisons,  and  when  there  is  a  diminished  supply  of 
oxygen — as  in  severe  and  continuous  dyspnoea,  poisoning  with  carbon 
monoxide,  hemorrhage,  etc.  In  these  cases  it  used  to  be  considered  that 
the  rise  in  the  excretion  of  nitrogen  was  due  to  an  increased  elimination  of 
urea,  because  no  exact  difference  was  made  between  the  quantity  of  urea 
and  of  total  nitrogen  in  the  urine.  Recent  researches  have  conclusively 
demonstrated  the  untrustworthiness  of  these  observations.  Since  PFLtJGER 
and  Borland  have  shown  that  16  per  cent  of  the  total  nitrogen  of  the  urine 
exists  under  physiological  conditions  in  other  compounds,  not  urea,  atten- 
tion has  been  called  to  the  relationship  of  the  different  nitrogenous  con- 
stituents of  the  urine  to  each  other,  and  it  has  been  found,  under  patho- 
logical conditions,  that  this  relationship  may  vary  considerably,  especially 
in  regard  to  the  urea.  We  have  numerous  determinations  by  different 
investigators,  such  as  Borland,  E.  Scrultze,  Camerer,  Voges,  Morner 
and  Sjoqvlst,  Gumlicr,  Bodtker,i  and  others,  on  the  relationship  of  the 
different  nitrogenous  constituents  to  each  other  in  the  normal  urine  of 
adults.  Sjoqvist  has  made  similar  determinations  on  new-born  babes 
from  1  to  7  days  old.  From  all  these  analyses  we  obtain  the  following 
figures  (A  for  adults  and  B  for  new-born  babes).  Of  the  total  nitrogen 
there  exists: 


>  Pflijger  and  Bohland,  Pfliiger's  Arch.,  38  and  43;  Bohland,  ibid.,  43;  Schultze, 
ihid.,  45;  Camerer,  Zeitschr.  f.  Biologic,  24,  2",  and  28;  Voges,  Ueber  die  Mischung 
der  stickstoffhaltigen  Bestandtheile  im  Ham,  etc.  (Inaiig.-Diss.  Berlin,  1892)  cited 
from  Maly's  Jahresber.,  22;  K.  Morner  and  Sjoqvist,  Skand.  Arch.  f.  Physiol.,  2. 
See  also  Sjoqvist,  Nord.  med.  Arkiv.  1892,  No.  36,  and  1894,  No.  10;  Gumlich,  Zeitschr. 
f.  physiol.  Chem.,  17;  Bodtker,  see  Maly's  Jahresber. ,  26. 


NITROGEN  OF    THE  URINE.  549 

A.  B. 

Per  Cent.  Per  Cent. 

Urea 84-91  73-76 

Ammonia 2-5  7 . 8-9 . 6 

Uric  acid 1-3  3.0-8.5 

Remaining  nitrogenous  substances  (extractives)..  .  .     7-12  7.3-14.7 

The  variable  relationship  between  uric  acid,  ammonia,  and  urea  nitrogen 
in  children  and  adults  is  remarkable,  since  the  urine  of  children  is  consider- 
ably richer  in  uric  acid  and  ammonia,  and  considerably  poorer  in  urea,  than 
the  urine  of  adults.  The  absolute  quantity  of  urea  nitrogen  in  adults 
amounts  to  about  10-16  grams  per  day.  In  disease  the  proportion  of  the 
nitrogenous  substances  may  be  markedly  changed,  and  a  decrease  in  the 
quantity  of  urea  and  an  increase  in  the  quantity  of  ammonia  have  been 
observed  in  certain  diseases  of  the  hver.  This  will  be  considered  in  detail  in 
connection  with  the  formation  of  urea  in  the  liver.  It  is  natural  that  there 
should  be  a  diminished  formation  of  urea  after  a  decrease  in  the  ingestion 
of  proteins  or  in  a  lowered  catabolism.  In  diseases  of  the  kidneys  which 
disturb  or  destroy  the  integrity  of  the  epithelium  of  the  convoluted  urinary 
passages,  the  ehmination  of  urea  is  considerably  diminished. 

Recently  by  means  of  Pfaux idler's  ^  method,  by  precipitating  the  urine 
with  phosphotungstic  acid  and  closely  studying  the  precipitate  as  well  as 
the  filtrate,  it  has  been  possible  to  learn  further  about  the  division  of  the 
nitrogen  of  the  urine.  We  determine  a,  the  total  nitrogen;  6,  the  nitrogen 
of  the  phosphotungstate  precipitate ;  and  c,  the  nitrogen  in  the  filtrate  from 
the  phosphotungstate  precipitate.  This  last  contains  the  urea,  hij^puric 
acid,  and  other  bodies  whose  nitrogen  is  ordinarily  designated  as  monamino- 
acid  nitrogen.  The  urea  nitrogen  is  especially  determined.  The  Ijodies 
precipitated  by  phosphotungstic  acid  are  not  all  known ;  but  uric  acid  and 
purine  bases,  ammonia,  creatinine,  pigments,  diamino-acids,  diamines  and 
ptomaines  (if  they  occur),  sulphocyanides,  carbamic  acid,  urine  mucoid, 
and  proteid  belong  to  this  group.  Of  these  bodies,  ammonia,  uric  acid, 
creatinine  and  jxirine  bases  are  specially  determined. 

The  urea  nitrogen  is  always  the  greatest  part  of  the  total  nitrogen,  but 
otherwise  the  division  of  the  nitrogen  undergoes  considerable  variation. 
According  to  v.  Jacksch^  normal  human  urine  contains  from  1.5  to  3 
per  cent  of  the  total  nitrogen  as  amino-acid  nitrogen  and  5.16  to  8.5  per 
cent  as  ammonia  and  purine  bodies.  Other  experimenters  have  obtained 
different  results,  and  our  knowledge  on  this  subject  is  not  sufficient.  Very 
great  variations  seem  to  occur  not  only  in  the  healthy  indi\'idual,  but  also 
and  to  a  greater  degree  in  diseased  conditions.^ 

'  Pfaundler,  Zeitschr.  f.  physiol.  Chem.,  30. 
^Zeitschr.  f.  klin.  Med.,  50. 

'See  Satta,  Hofmeister's  Beitrage,  6,  which  also  gives  the  literature,  and  Erben, 
Zeitschr.  f.  Heilkunde,  25. 


550  URINE. 

Formation  of  Urea  in  the  Organism.  The  experiments  to  produce  urea 
directly  from  proteins  by  oxidation  have  led  to  the  formation  of  some  guan- 
idine,  but  urea  has  not  been  obtained  positively.  On  the  hydrolysis  of 
proteins  arginine  has  been  found  among  other  products,  and  as  it  is  also 
produced  in  trv^ptic  digestion,  it  is  possible  that  a  small  portion  of  the  urea 
is  produced  in  this  manner,  varying  according  to  the  kind  of  protein 
(Drechsel,  Kossel,  see  Chapter  II).  Drechsel  claims  that  about  10  per 
cent  of  the  urea  can  be  accounted  for  in  this  way. 

The  possibility  of  a  formation  of  urea  from  arginine  has  gained  in  in- 
terest since  Kossel  and  Da  kin  have  discovered  the  presence  of  an  enzyme, 
arginase,  in  the  liver  and  other  orgaas,  which  has  the  power  of  splitting 
arginine  with  the  formation  of  urea.  Thompson^  has  recently  given  a  direct 
proof  for  the  formation  of  urea  from  arginine.  The  introduction  of  arginine 
into  the  body  of  a  dog  either  per  os  or  subcutaneously  has  in  liis  experi- 
ments led  to  an  elimination  of  urea.  While  outside  of  the  body  only  one- 
half  of  the  nitrogen  of  arginine  is  split  off  as  urea  and  the  other  half  as 
ornithine,  in  the  above  experiments  the  increase  in  urea  in  several  instances 
corresponded  to  the  greater  part  if  not  the  w^hole  of  the  nitrogen  of  the 
arginine  introduced.  In  these  cases,  wdthout  mentioning  that  the  arginine 
seemed  to  raise  the  nitrogen  catabolism,  probably  also  urea  was  formed 
from  the  ornithine.  This  can  be  explained  Ijy  a  deamidation  of  the  orni- 
thine and  formation  of  urea  from  the  ammonia  and  carbon  dioxide  split  off. 

By  the  action  of  alkalies,  as  above  mentioned  (Chapter  XI),  urea  may 
be  formed  from  creatine;  still  such  an  origin  of  urea  in  the  animal  body 
has  not  thus  far  been  proved. 

The  amino-acids  are  considered  as  special  mother-substances  of  urea. 
By  the  researches  of  Schultzex  and  Nencki  and  Salkowski  wath  leucine 
and  glycocoll,  those  of  Stolte  with  several  amino-acids,  and  those  of  v. 
Knieriem  with  asparagine.  it  has  been  shown  that  the  amino-acids  are  in 
part  converted  into  urea  in  the  animal  organism.  The  investigations  by 
Salaskin  with  the  three  amino-acids,  glycocoll,  leucine,  and  aspartic  acid, 
have  unmistakably  shown  that  the  surviving  dog-liver,  supplied  with  arterial 
blood,  has  the  property  of  transforming  the  above  amino-acids  into  urea 
or  a  closely  allied  substance.  The  researches  of  Loewi  with  the  "urea- 
forming  "  enzyme  of  the  liver,  discovered  by  Richet,  and  glycocoll  or 
leucine,  as  also  the  researches  of  Ascoli,^  have  led  to  similar  results,  but 
it  must  be  remarked  that  we  have  no  proof  as  to  the  identity  of  the  newly 

*  Kossel  and  Dakin.  Zeitschr.  f.  physiol.  Chem.,  41;  Thompson,  Journ.  of  Physiol., 
32  and  33. 

^  Schultzen  and  Nencki,  Zeitschr.  f.  Biologic,  8;  v.  Knieriem,  ibid.,  10;  Salkowski, 
Zeitschr.  f.  physiol.  Chem.,  4;  Salaskin,  ibid.,  25;  Loewi,  ibid.,  2o;  Stolte,  Hofmeistcr's 
Beitrage,  5;  Richet,  Compt.  rend.,  118,  and  Compt.  rend.  Soc.  biol.,  49;  Ascoli, 
Pfluger's  Arch.,  72. 


FORMATION   OF  UREA.  551 

formed  substance  with  urea.  Xothing  can  be  stated  in  regard  to  the  extent 
of  formation  of  amino-acids  in  the  physiological  destruction  of  proteins  in 
the  animal  body,  with  the  exception  of  those  formed  in  the  intestinal  diges- 
tion. The  possibihty  of  such  a  formation  of  urea  is  beyond  dispute.  As 
sho\^-n  by  Abderhaldex  ^  with  Teruughi  and  Babkix,  the  polypeptides, 
like  the  amino-acids,  can  also  be  converted  into  urea  in  the  animal  body. 

Nothing  positive  can  be  said  in  regard  to  the  manner  in  which  this 
formation  of  urea  occurs;  but  it  is  admitted  that  it  is  partly  a  formation 
from  ammonia  and  partly  from  carbamic  acid. 

The  possibilit}-  of  a  formation  of  urea  from  ammonia  has  been  positively 
shown.  Thus  the  researches  of  v.  Kxieriem.  Salkowski.  Feder,  I.  Muxk, 
CoRAXDA,  SchmiedeberCt  and  Fr.  Walter.  Hallervordex,  and  Pohl 
and  }>^iJxzER,2  on  the  behavior  of  ammonium  salts  in  the  animal  body  and 
the  ehmination  of  the  ammonia  imder  various  conditions,  have  shown 
that  not  only  ammonium  carbonate,  but  also  those  ammonium  salts  wliich 
are  burnt  into  carbonate  in  the  organism,  are  transformed  into  urea  by 
carnivora  as  well  as  herbivora.  v.  Schroeder.^  by  irrigating  the  sur\-i\-ing 
dog's  liver  with  blood  treated  with  ammonium  carbonate  or  ammonium 
formate,  has  shown  that  the  formation  of  urea  takes  place,  at  least  in  part, 
in  this  organ.  Nexcki,  Pawlow,  Zaleski  and  Salaskix  *  have  also  found 
that  in  dogs  the  quantity  of  ammonia  in  the  blood  from  the  portal  vein  is 
considerably  greater  than  that  from  the  hepatic  vein,  and  they  claim  that 
the  Hver  retains  in  great  part  the  ammonia  thus  supplied.  The  formation 
of  urea  from  ammonia  in  the  liver  is  a  positively  proved  fact,  and  the  urea 
formation  from  ammonium  carbonate  is  to  be  considered  as  a  synthesis 
with  the  elimination  of  water. 

The  assumption  of  a  spUtting  off  of  ammonia  from  amino-acids  is  not 
difficult  of  conception,  as  now,  especially  from  the  investigations  men- 
tioned in  Chapter  VIII.  we  know  T\-ith  positiveness  that  deamidation  of 
amino-acids  does  take  place  in  the  animal  bod}'.  The  ammonia  spUt  off 
finds  in  the  blood  and  tissues  the  carbon  dioxide  necessary-  for  the  formation 
of  carbonate,  and  to  all  appearances  the  conditions  are  also  suitable  for 
the  formation  of  carbamate. 

Important  observations  have  been  made  wliich  give  support  to  the  ^iews 
of  ScHULTZEX'  and  Nexcki.^  namely,  that  the  amino-acids  are  transformed 

'Zeitschr.  f.  physiol.  Chem.,  4". 

''v.  Knieriem,  Zeitschr.  f.  Biolotrie,  10;  Feder,  ibiyl.,  13;  Salkowski,  Zeitschr.  f. 
Biologie,  1;  Munk,  ibid.,  2;  Coranda,  Arch.  f.  exp.  Path.  u.  Pharm.,  12;  Scjimiede- 
berg  and  Walter,  ibid.,  7;  Hallerv'orden,  ibid.,  10;  Pohl  and  Miinzer,  Arch.  f.  exp. 
Path.  u.  Phann.,  43. 

^  Arch.  f.  exp.  Path.  u.  Pharm.,  15.     See  al.^o  Salomon,  Virchow's  Arch.,  97. 

*  Arch,  des  sciences  bid.  do  St.  Petersbourg,  4;  see  also  Chapter  \'T,  p.  241. 

*  Zeitschr.  f .  Biologie,  8. 


552  URINE. 

into  urea  with  carbamic  acid  as  an  intermediate  step.  Drechsel  has 
shown  that  the  amino-acids  yield  carbamic  acid  by  oxidation  in  alkaline 
fluid  outside  of  the  organism,  and  he  obtained  urea  from  ammonium  car- 
bamate by  passing  an  alternating  electric  current  through  its  solution, 
i.e.,  by  alternate  oxidation  and  reduction.  Drechsel  has  also  been 
able  to  detect  small  quantities  of  carbamates  in  blood,  and  later  in  con- 
junction with  Abel  he  detected  carbamic  acid  in  alkaline  horse's  urine. 
Drechsel  therefore  accepts  the  formation  of  urea  from  ammonium  car- 
bamate, and  according  to  him  the  alternating  oxidation  and  reduction  take 
place  in  the  following  way: 

H4N.O.CO.NH2  +  0  =  H2N.O.CO.NH2  +  H20 

Ammonium  carbamate 

H2N.O.CO.NH2  +  H2  =  H2N.CO.NH2  +  H2O. 

Urea 

Abel  and  Muirhead  1  have  later  observed  an  abundant  eUmination  of 
carbamic  acid  in  human  and  dog's  urine  after  the  administration  of  large 
quantities  of  milk  of  lime,  and  the  probaljility  of  the  regular  appearance  of 
this  acid  in  normal  acid-reacting  human  and  dog's  urine  has  been  demon- 
strated by  M.  Nencki  and  Hahn.^  These  last-mentioned  investigators 
have  also  given  very  important  support  to  the  theory  of  the  formation  of 
urea  from  ammonium  carbamate  by  observations  on  dogs  with  Eck's 
fistula.  In  this  case  the  portal  vein  is  directly  connected  with  the  inferior 
vena  cava,  and  communication  is  thus  established  between  the  two,  so  that 
the  blood  of  the  portal  vein  flows  directly  into  the  vena  cava,  without 
passing  through  the  liver.  Nencki  and  Hahn  observed  violent  symptoms 
of  poisoning  in  dogs  fed  on  meat  and  operated  upon  by  Pawlow  and 
Massen,  and  these  symptoms  were  quite  identical  with  those  obtained  on 
introducing  carbamate  into  the  blood.^  These  symptoms  also  appear 
after  the  introduction  of  carbamate  into  the  stomach,  while  the  introduc- 
tion of  carbamate  into  the  stomach  of  a  normal  dog  had  no  action.  As 
these  observers  also  found  that  the  urine  of  the  dog  on  w^hich  the  operation 
was  made  was  richer  in  carbamate  than  that  of  the  normal  dog,  they  con- 
cluded that  the  symptoms  were  due  to  the  non-transformation  of  the 
ammonium  carbamate  into  urea  in  the  liver,  and  they  consider  the  amnio- 

'  Drechsel,  Ber.  d.  siichs.  Gesellsch.  d.  Wissensch*. ,  1875.  See  also  Journ.  f.  prakt. 
Chem.  (N.  F  ),  12,  16,  and  22;  Abel,  Arch,  f  (Anat.  u.)  Physiol.,  1891;  Abel  and 
Muirhead,  Arch.  f.  exp  Path.  u.  Pharm  ,  31. 

''  Hahn,  Massen,  Nencki  et  Pawlow,  La  fi.stule  d'  Eck  de  la  veine  cave  inf^rieure  et 
de  la  veine  porte,  etc      Arch,  des  sciences  biol.  de  St,  Petersbourg,  1,  No.  4,  1892. 

''  Rothberger  and  Winterberg,  Zeitschr  f.  exp.  Path,  u  Therap  .  1,  have  found  that 
the  phenomena  of  meat  poisoning  and  the  carbamic-acid  intoxication  are  not  identical. 


FORMATION  OF   UREA.  553 

nium  carbamate  as  the  substance  from  which  the  urea  is  derived  in  the 
mammahan  liver. 

The  view  as  to  the  formation  of  urea  from  ammonium  carbamate  does 
not  contradict  the  above  statement  as  to  the  transformation  of  the  carbonate 
into  urea,  since  we  can  imagine  that  the  carl^onate  is  first  converted  into 
carbamate  with  the  expulsion  of  a  molecule  of  water,  and  that  this  then  is 
transformed  into  urea  with  the  expulsion  of  a  second  molecule  of  water. 

F.  HoFMEiSTER  ^  has  found  in  the  oxidation  of  different  members  of  the 
fat  series,  as  well  as  in  amino-acids  and  proteins,  that  urea  was  formed 
in  the  presence  of  ammonia,  and  he  therefore  suggests  the  possibility  that 
urea  may  be  formed  by  an  oxidation-synthesis.  According  to  him,  in  the 
oxidation  of  nitrogenous  substances  a  radical  CONH2,  containing  the 
amide  group,  unites  at  the  moment  of  formation  with  the  radical  NH2 
remaining  on  the  oxidation  of  ammonia,  forming  urea. 

Besides  the  above-mentioned  theories  as  to  the  formation  of  urea,  there 
are  others  which  will  not  be  given,  because  the  only  theory  which  has  thus 
far  been  positively  demonstrated  is  the  formation  of  urea  from  ammonium 
compounds  and  amino-acids  in  the  liver. 

The  liver  is  the  only  organ  in  which,  up  to  the  present  time,  a  formation 
of  urea  has  been  directh"  detected '?  and  the  question  arises,  what  importance 
has  this  urea  formation  which  takes  place  in  the  liver?  Is  the  urea  wholly 
or  chiefly  formed  in  the  liver? 

If  the  liver  is  the  only  organ  capable  of  forming  urea,  it  is  to  be  expected, 
on  the  extirpation  or  atrophy  of  that  organ,  that  a  reduced  or,  in  short  experi- 
ments, at  least  a  strongly  diminished  elimination  of  urea  should  occur.  As 
at  least  a  part  of  the  urea  is  formed  in  the  liver  from  ammonium  com- 
pounds, a  simultaneous  increase  in  the  elimination  of  ammonia  is  to  be 
expected. 

The  extirpation  and  atrophy  experiments  made  on  animals  by  different, 
methods  by  Nexcki  and  Hahx,  Slosse.  Liebleix.  Nexcki  and  Pawlow 
Salaskin  and  Zaleski  ^  have  shown  that  sometimes  a  rather  marked  in- 
crease of  ammonia  and  a  diminished  elimination  of  urea  takes  place  after 
the  operation,  but  also  that  there  are  cases  in  which,  irrespective  of  the 
pronounced  atrophy,  an  alnmdant  formation  of  urea  occurs,  and  no  appre- 

'  Arch.  f.  exp.  Path.  u.  Pharni.,  37. 

'  In  regard  to  the  investigations  of  Prevost  and  Dumas,  Meissner,  Volt,  Grehant. 
Gscheidlen  and  Salkowski,  and  others,  on  the  role  of  the  kidneys  in  the  formation  of 
urea,  see  v.  Schroeder,  Arch.  f.  exp.  Path.  u.  Pharm.,  15  and  19,  and  Voit,  Zeitschr. 
f.  Biologie,  4. 

•*  Nencki  and  Hahn,  1.  c;  Slosse,  Arch.  f.  (Anat.  u.)  Physiol.,  1890;  Lieblein,  Arch. 
f.  exp.  Path.  u.  Pharm..  33;  Nencki  and  Pawlow,  Arch,  des  scienc.  biol.  de  St.  Pdters- 
bouTg.  o.  See  also  v.  .Meister,  Maly's  Jahresbr.,  25;  Salaskin  and  Zaleski,  Zeitschr.  f. 
physiol.  Chcm.,  29. 


554  URINE. 

ciable,  if  any,  change  in  the  proportion  of  ammonia  to  the  total  nitrogen 
and  urea  is  observed.  After  shutting  out  tlie  organs  of  the  posterior  part 
of  the  body,  especially  the  hver  and  kidneys,  from  the  circulation,  Kauf- 
MANN  ^  also  found  an  important  increase  in  the  urea  of  the  blood,  and  these 
different  observations  show  that  the  liver  is  not  the  only  organ,  in  the 
various  animals  experimented  upon,  in  which  urea  is  formed. 

The  observations  made  by  numerous  investigators  ^  on  human  beings 
with  cirrhosis  of  the  liver,  acute  yellow  atrophy  of  the  liver,  and  phosphorus 
poisoning  have  led  to  the  same  result.  These  investigations  teach  that  in 
certain  cases  the  proportion  of  the  nitrogenous  substances  may  be  so 
changed  that  urea  is  only  50-60  per  cent  of  the  total  nitrogen,  while  in  other 
cases,  on  the  contrary,  even  in  very  extensive  atrophy  of  the  liver-cells, 
the  formation  of  urea  is  not  diminished,  neither  is  the  proportion  between 
the  total  nitrogen,  urea,  and  ammonia  essentially  changed.  Even  in  the 
cases  in  which  the  formation  of  urea  was  relatively  diminished  and  the 
elimination  of  ammonia  considerably  increased  further  investigation  must 
be  instituted  before  it  will  be  possible  to  assume  a  reduced  ability  of  the 
organism  to  produce  urea.  An  increased  elimination  of  ammonia  may,  as 
shown  by  ^Iijnzer  in  the  case  of  acute  phosphorus  poisoning,  be  dependent 
upon  the  formation  of  abnormally  large  quantities  of  acids,  caused  by  ab- 
normal metaliolism,  and  these  acids  require  a  greater  quantit}"^  of  ammonia 
for  their  neutralization  according  to  the  law  of  elimination  of  ammonia, 
which  will  be  given  later.  That  an  abnormal  formation  of  acid  occurs 
after  the  cutting  out  of  the  Hver  has  been  especially  shown  by  Salaskin  and 
Zaleski.3 

•  For  the  present  we  are  not  justified  in  the  statement  that  the  liver  is 
the  only  organ  in  which  urea  is  formed,  and  only  continued  investigation 
can  yield  further  information  as  to  the  extent  and  importance  of  the  forma- 
tion of  urea  in  the  liver  from  ammonium  compounds. 

Properties  and  Reactions  of  Urea.  Urea  crystallizes  in  needles  or  in 
long,  colorless,  four-sided,  often  hollow,  anhydrous  rhombic  prisms.  It  has 
a  neutral  reaction,  and  produces  a  cooling  sensation  on  the  tongue  like  salt- 
peter. It  melts  at  132°  C.  At  ordinary  tem]ieratures  it  dissolves  in  an  equal 
weight  of  water  and  in  five  parts  alcohol ;  it  requires  one  part  boiling  alcohol 
for  solution;  it  is  insoluble  in  alcohol-free  anhydrous  ether,  and  also  in 
chloroform.     If  urea  in  substance  is  heated  in  a  test-tube,  it  melts,  decom- 

'  Compt.  rend.  soc.  biol.,  46,  and  Arch,  de  Physiol.  (5),  6. 

^  See  Hallervorden,  Arch,  f .  exp.  Path.  u.  Pharm.,  12;  Weintraud,  ibid.,  31 ;  Miinzer 
and  Winterberg,  ibid.,  33;  Sta 'elman'i,  Deiitsch.  Arch,  f .  klin.  Med.,  33;  Fawitzki, 
ibid.,  45;  Miinzer,  ibid.,  52;  Frankel,  Berlin,  klin.  Wochenschr.,  1878;  Richtcr,  ibid., 
1896;  Morner  and  Sjoqvist,  Skand.  Arch.  f.  Physiol.,  2,  and  Sj6.-]vist,  Nord-  l]ed. 
Arkiv,  1892;  Gumlich,  Zeitschr.  f.  physiol.  Chem.,  17;  v.  Noorden,  Lehrb.  d.  Pathol, 
des  Stoffwechsels,  2.  Aufl.,  Bd.  1,  104. 

^Zeitschr.  f.  physiol.  Chem.,  29. 


PROPERTIES   OF  UREA,  o55 

poses,  gives  off  ammonia,  and  leaves  finally  a  non-transparent  white  residue 
which,  among  other  substances,  contains  cyanuric  acid  and  biuret,  which 
latter  dissolves  in  water,  giving  a  beautiful  reddish-violet  Uciuid  with  copper 
sulphate  and  alkali  {biuret  reaction).  On  heating  with  baryta-water  or 
caustic  allcah.  also  in  the  so-called  alkaline  fermentation  of  urine  caused  by 
micro-organisms,  urea  sphts  into  carbon  dioxide  and  ammonia  with  the 
addition  of  water.  The  same  decomposition  products  are  produced  when 
urea  is  heated  with  concentrated  sulphuric  acid.  An  alkaUne  solution  of 
sodium  hypobromite  decomposes  urea  into  nitrogen,  carbon  dioxide,  and 
water  according  to  the  equation 

CON2H4  +  SNaOBr = 3NaBr  +  CO2  +  2H2O  +  Ng. 

With  a  concentrated  solution  of  furfurol  and  hydrochloric  acid  urea 
in  substance  gives  a  coloration  passing  from  yellow,  green,  blue,  to  violet, 
and  then  beautiful  purple-violet  after  a  few  minutes  (Schiff's  reaction). 
According  to  Huppert  ^  the  test  is  best  performed  by  taking  2  c.c.  of  a 
concentrated  furfurol  solution,  4-6  drops  of  concentrated  hydrochloric  acid, 
and  adding  to  this  mixture,  which  must  not  be  red,  a  small  crj'stal  of  urea. 
A  deep  violet  coloration  appears  in  a  few  minutes. 

Urea  forms  cr3-stanine  compounds  with  many  acids.  Among  these  the 
one  with  nitric  acid  and  the  one  with  oxahc  acid  are  the  most  important. 

Urea  Nitrate,  C0(NH2)2-HN03.  On  crj-stalhzing  cjuickly  tliis  com- 
pound forms  thin  rhombic  or  six-sided  overlapping  tiles,  or  colorless 
plates,  with  an  angle  of  82°.  When  crj'stalhzing  slowly,  larger  and 
thicker  rhombic  pillars  or  plates  are  obtained.  Tliis  compound  is  rather 
easily  soluble  in  pure  water,  but  is  considerably  less  soluble  in  water  con 
taining  nitric  acid;  it  may  be  obtained  by  treating  a  concentrated  solutiori 
of  urea  with  an  excess  of  strong  nitric  acid  free  from  nitrous  acid.  On 
heating  this  compound  it  volatilizes  without  leaA^ng  a  residue. 

This  compound  may  be  employed  with  advantage  in  detecting  small  amounts 
of  urea.  A  drop  of  the  concentrated  solution  is  placed  on  a  microscope-slide  and 
the  cover-glass  j^laced  upon  it;  a  drop  of  nitric  acid  is  then  placed  on  the  side 
of  the  cover-glass  and  allowed  to  flow  under.  The  formation  of  crystals  begins 
•where  the  solution  and  the  nitric  acid  meet.  Alkali  nitrates  may  crystallize 
very  similarly  to  urea  nitrate  when  they  are  contaminated  with  other  bodies : 
therefore,  in  testing  for  urea,  the  cr}'stals  must  be  identified  as  m-ea  nitrate  by 
heating  and  by  other  means. 

Urea  Ox-\late,  2. CO (NHo) 2 -1120204.  This  compound  is  more  spar- 
ingly soluble  in  water  than  the  nitric-acid  compound.  It  is  obtained  in 
rhombic  or  six-sided  prisms  or  plates  on  adding  a  saturated  oxalic-acid 
solution  to  a  concentrated  sokition  of  urea. 


'  Huppert-Xeubaucr,  Analyse  dcs  Hams,  10.  Aufl.,  296. 


556  URINE. 

Urea  also  forms  combinations  with  mercuric  nitrate  in  variable  propor- 
tions. If  a  ver}'  faintly  acid  mercuric-nitrate  solution  is  added  to  a  2  per 
cent  solution  of  urea  and  the  mixture  carefully  neutralized,  a  compound  is 
obtained  of  a  constant  composition  which  contains  for  every  10  parts  of 
urea  72  parts  of  mercuric  oxide.  This  compound  serves  as  the  basis  of 
Liebig's  titration  method.  Urea  combines  also  with  salts,  forming  mostly 
crystalUzable  combinations,  as,  for  instance,  with  sodium  chloride,  with 
the  chlorides  of  the  heav}^  metals,  etc.  An  alkaline  but  not  a  neutral 
solution  of  urea  is  precipitated  with  mercuric  chloride. 

*  If  urea  is  dissolved  in  dilute  hydrochloric  acid  and  then  an  excess  of  formal- 
dehyde is  added,  a  thick,  white,  granular  precipitate  is  obtained  which  is  difficultly 
soluble  and  whose  composition  is  somewhat  disputed.^  With  phenylhj^drazine, 
urea  in  strong  acetic  acid  gives  a  colorless  crystalline  compound  of  phenyl- 
semicarbazid,  CcHsNH.NH.C0NH2,  which  is  soluble  with  difficulty  in  cold  water 
and  melts  at  172°  C.  (Jaffe  '). 

The  method  of  preparing  urea  from  urine  is  in  the  main  as  follows:  Con- 
centrate the  urine,  which  has  been  faintl}'  acidified  with  sulphuric  acid,  at  a 
low  temperature,  add  an  excess  of  nitric  acid,  at  the  same  time  keeping  the 
mixture  cool,  press  the  precipitate  well,  decompose  it  in  water  with  freshly 
precipitated  barium  carbonate,  dry  on  the  water-bath,  extract  the  residue 
with  strong  alcohol,  decolorize  when  necessary  with  animal  charcoal,  and 
filter  while  warm.  The  urea  which  crystallizes  on  cooling  is  purified  by 
recrystallization  from  warm  alcohol.  A  further  quantity  of  urea  may  be 
obtained  from  the  mother-liquor  by  concentration.  The  urea  is  purified 
from  contaminating  mineral  bodies  by  redissolving  in  alcohol-ether.  If  it 
is  only  necessary  to  detect  the  presence  of  urea  in  urine,  it  is  sufficient  to 
concentrate  a  little  of  the  urine  on  a  watch-glass  and,  after  cooling,  treat  it 
with  an  excess  of  nitric  acid.     In  this  way  we  obtain  crystals  of  urea  nitrate. 

Quantitative  Estimation  of  the  Total  Nitrogen  and  Urea  in  Urine.  Among 
the  various  methods  proposed  for  the  estimation  of  the  total  nitrogen,  that 
suggested  by  Kjeldahl  is  to  be  recommended.  But  as  Liebig's  method 
for  the  estimation  of  urea  is  really  a  method  for  determining  the  total 
nitrogen,  and  as  the  physician  has  not  always  at  hand  the  apparatus  and 
utensils  necessary  for  a  Kjeldahl  determination,  he  often  makes  use  of 
this  method ;  hence  both  wnll  be  given  in  detail. 

Kjeldahl's  method  consists  in  transforming  all  the  nitrogen  of  the 
organic  substances  into  ammonia  by  heating  with  a  sufficiently  concentrated 
sulphuric  acid.  The  ammonia  is  distilled  off  after  supersaturating  with 
alkali  and  the  ammonia  collected  in  standard  sulphuric  acid.  The  follow- 
ing reagents  are  necessary' : 

1.  Sulphuric  Acid.  Either  a  mixture  of  equal  volumes  of  pure  concen- 
trated and  fuming  sulphuric  acid  or  else  a  solution  of  200  grams  phosphoric 

^  See  Tollens  and  his  pupils,  Bcr.   d.  doutsch.  chem.  Gescllsch.,  29,  2751;  Gold- 
schmidt,  ibid. ,2^,  and  Chcm.  Centralbl.,  1897,  1,  33;  Thoms,  ibid.,  2,  144  and  737. 
^  Zeitschr.  f.  physiol.  Chem.,  22. 


ESTIMATION  OF  UREA.  557 

anhydride  in  1  liter  of  pure  concentrated  sulphuric  acid.  2.  Caustic  soda  free 
from  nitrates,  30-40  per  cent  solution.  The  quantity  of  this  caustic-soda 
solution  necessary  to  neutralize  10  c.c.  of  the  acid  mixture  must  be  deter- 
mined. 3.  Metallic  mercury  or  pure  yellow  mercuric  oxide.  (The  addition  of 
this  facilitates  the  destiTiction  of  the  organic  substances.)  4.  A  potassium- 
sulphide  solution  of  4  per  cent,  whose  object  is  to  decompose  any  mercuric 
amide  combination  which  might  not  evolve  its  ammonia  completely  during 
the  distillation  with  caustic  soda.  5.  N/5  sulphuric  acid  and  N/5  caustic- 
soda  solution. 

In  performing  the  determination  5  c.c.  of  the  carefully  measured  and 
filtered  urine  is  placed  in  a  long-neck  Kjeldahl  flask,  a  drop  of  mercury  or 
about  0.3  gram  of  mercuric  oxide  added,  and  then  treated  with  10-15  c.c. 
of  the  strong  sulphuric  acid.  The  contents  are  heated  very  carefully, 
placing  the  flask  at  an  angle,  until  they  just  begin  to  boil  gently ;  this  is 
continued  for  about  half  an  hour  after  the  mixture  becomes  colorless.  On 
coohng  the  contents  are  transferred  to  a  voluminous  distilling-flask,  care- 
fully washing  the  Kjeldahl  flask  with  water,  and  the  greater  part  of  the  acid 
is  neutralized  by  caustic  soda.  A  few  zinc  shavings  are  added  to  prevent 
too  rapid  ebullition  on  distillation,  and  then  an  excess  of  caustic-soda  solu- 
tion which  has  previously  been  treated  with  30-40  c.c.  of  the  potassium- 
sulphide  solution.  The  flask  is  quickly  connected  with  the  condenser-tube 
and  all  the  ammonia  distilled  off".  In  order  to  prevent  loss  of  ammonia  it  is 
best  to  lower  the  end  of  the  exit-tube  below  the  surface  of  the  acid,  and  the 
regurgitation  of  the  acid  is  prevented  by  having  a  Ijulb  Ijlown  on  the  exit- 
tube.  Not  less  than  25-30  c.c.  of  the  standard  acid  is  used  for  every  5  c.c. 
of  urine,  and  on  completion  of  the  distillation  the  acid  is  retitrated  with 
N/5  caustic  soda,  using  rosolic  acid,  tincture  of  cochineal,  or  lacmoid 
as  indicator.  Each  cubic  centimeter  of  the  acid  corresponds  to  2.8 
milligrams  nitrogen.  As  a  control  and  in  order  to  test  the  purity  of  the 
reagents,  or  to  eliminate  any  error  caused  by  an  accidental  quantity  of 
ammonia  in  the  air,  we  always  make  a  blank  determination  with  the 
reagents. 

Liebig's  method  is  based  upon  the  fact  that  a  dilute  solution  of  mer- 
curic nitrate  under  proper  conditions  precipitates  all  the  urea  from  its 
solution,  forming  a  compound  of  constant  composition.  As  indicator,  a 
soda  solution  or  a  thin  paste  of  sodium  bicarbonate  is  used.  An  excess  of 
mercuric  nitrate  produces  herewith  a  yellow  or  yellowish-brown  compound, 
while  the  compound  of  urea  and  mercury  is  white.  PFLtJuER  ^  has  given 
full  particulars  for  this  method;  therefore  we  will  describe  Pflijger's 
modification  of  Liebig's  method. 


Pfliiger,  and  Pfliiger  and  Bohland,  in  I  Auger's  Arch.  21, 36,  37,  and  40. 


558  URINE. 

As  phosphoric  acid  is  also  precipitated  by  the  mercuric-nitrate  solution,  this 
must  be  removed  from  the  urine  by  the  addition  of  a  baryta  solution  before  titra- 
tion. Pfluger  also  suggested  that  the  acidity  produced  by  the  mercury  solution 
be  neutralized  during  titration  by  the  addition  of  a  soda  solution.  The  liquids 
necessary  for  the  titration  are  the  following: 

1.  M crcuric-nitratc  Solution.  This  solution  is  calculated  for  a  2  per  cent  urea 
solution,  and  20  c.c.  of  the  first  should  correspond  to  10  c.c.  of  the  latter.  Each 
cubic  centimeter  of  the  mercury  solution  corresponds  to  0.01  gram  urea.  As  a 
small  excess  of  HgO  is  necessary  in  the  urine  to  cause  the  final  reaction  (with 
alkali  carbonate  or  bicarbonate)  to  appear,  each  cubic  centimeter  of  the  mercury 
solution  must  contain  0.0772  instead  of  0.0720  gram  HgO.  The  mercury  solution 
contains  therefore  77.2  grams  HgO  in  1  liter. 

The  solution  may  be  prepared  from  pure  mercury  or  mercuric  oxide  by  dis- 
solving in  nitric  acid.  The  solution,  freed  as  completely  as  possible  from  an 
excess  of  acid,  is  diluted  by  the  careful  addition  of  water,  stirring  meanwhile, 
until  it  has  a  specific  gravity  of  1.10,  or  a  little  higher,  at  20°  C.  The  solution 
is  standardized  with  a  2  per  cent  solution  of  pure  urea  which  has  been  dried  over 
sulphuric  acid  and  the  operation  conducted  as  will  be  described  later.  If  the 
solution  is  too  concentrated,  it  is  corrected  by  carefully  adding  the  necessary 
amount  of  water,  avoiding  the  precipitation  of  the  basic  salt,  and  titrating  again. 
The  solution  is  correct  if  19.8  c.c.  of  it,  added  at  once  to  10  c.c  of  the  urea  solution 
and  the  quantity  (11-12  c.c.  or  more)  of  normal  soda  solution  necessary  to  nearly 
completely  neutralize  the  liquid,  gives  the  final  reaction  when  exactly  20  c.c.  of 
the  mercury  solution  has  been  employed. 

2.  Baryta  Solution.  This  consists  of  1  vol.  of  barium  nitrate  and  2  vols,  of 
barium-hydrate  solution,  both  saturated  at  the  ordinary  temperature. 

3.  Normal  Soda  Solution.  This  solution  contains  53  grams  of  pure  anhydrous 
sodium  carbonate  in  1  liter  of  water.  According  to  PFLiJGER  a  solution  having 
a  specific  gravity  of  1.053  is  sufficient.  The  amount  of  this  soda  solution  neces- 
sary to  completely  neutralize  the  acid  set  free  during  the  titration  is  determined 
by  titrating  with  a  pure  2  per  cent  urea  solution.  To  facilitate  operations  a  table 
can  be  made  showing  the  cjuantity  of  soda  solution  necessary  when  from  10  to 
35  c.c.  of  the  mercmy  solution  is  used. 

Before  the  titration  the  following  must  be  considered.  The  chlorides  of 
the  urine  interfere  with  the  titration  in  that  a  part  of  the  mercuric  nitrate 
is  transformed  into  mercuric  chloride,  which  does  not  precipitate  the  urea. 
The  chlorides  of  the  urine  are  therefore  removed  by  a  silver-nitrate  solu- 
tion, which  also  removes  any  bromine  or  iodine  compounds  which  may 
exist  in  the  urine.  If  the  urine  contains  proteid  in  noticeable  amounts,  it 
must  be  removed  by  coagulation  and  the  addition  of  acetic  acid,  but  care 
must  be  taken  that  the  concentration  and  the  volume  of  the  urine  are  not 
changed  during  these  operations.  If  the  urine  contains  ammonium  car- 
bonate in  noticeable  quantities,  caused  by  alkaline  fermentation,  this  titra- 
tion method  cannot  be  applied.  The  same  is  true  of  urine  containing 
leucine,  tyrosine,  or  medicinal  preparations  precipitated  by  mercuric 
nitrate. 

In  cases  where  the  urine  is  free  from  proteid  er  sugar  and  not  specially 
poor  in  chlorides,  the  quantity  of  urea,  and  also  the  approximate  quantity 
of  mercuric  nitrate  necessary  for  the  titration,  may  be  learned  from  the 
specific  gravity.     A  specific  gravity  of  1.010  corresponds  to  about  10  p.  m., 


ESTIMATION  OF  UREA.  559 

a  specific  gravity  of  1.015  generally  somewhat  less  than  15  p.  m.,  and  a 
specific  gravity  of  1.015-1.020  about  15-20  p.  m.  urea.  With  a  specific 
gravity  higher  than  1.020  the  urine  generally  contains  more  than  20  p.  m, 
of  urea,  and  above  this  point  the  amount  of  urea  increases  much  more 
rapidly  than  the  specific  gravity,  so  that  with  a  specific  gravity  of  1.030  it 
contains  over  40  p.  m.  urea.  Fever-urines  with  a  specific  gravity  above 
1.020  sometimes  contain  30-40  p.  m.  urea,  or  even  more. 

Preparation  for  the  Titration.  If  a  large  amount  of  urea  is  sus- 
pected from  a  high  specific  gravity,  the  urine  must  first  he  diluted  with  a 
carefully  measured  quantity  of  water,  so  that  the  amount  of  urea  is  re- 
duced below  30  p.  m.  In  a  special  portion  of  the  same  urine  the  amount  of 
chlorides  is  determined  by  one  of  the  methods  w^hich  will  1  )e  given  later,  and 
the  number  of  cubic  centimeters  of  silver-nitrate  solution  necessarj-  is 
noted.  Then  a  larger  quantity  of  urine,  say  100  c.c,  is  mixed  with  one- 
half  or,  if  this  is  not  sufficient  to  precipitate  all  the  sulphuric  and  phos- 
phoric acids,  with  an  equal  volume  of  the  baryta  solution;  it  is  then  allowed 
to  stand  a  little  while,  and  the  precipitate  is  filtered  through  a  dried  filter. 
From  the  filtrate  containing  the  urine  diluted  with  water  a  proper  quan- 
tity, corresponding  to  about  60  c.c.  of  the  original  urine,  is  measured,  and 
exactly  neutralized  with  nitric  acid  added  from  a  l^urette,  so  that  the  exact 
quantity  employed  is  known.  The  neutralized  mixture  of  urine  and  barj^ta 
is  treated  with  the  proper  quantity  of  silver-nitrate  solution  necessary  to 
completely  precipitate  the  clilorides,  which  were  ascertained  by  a  previous 
determination.  Tlie  mixture,  containing  a  known  volume  of  urine,  is  now 
filtered  through  a  dried  filter  into  a  flask,  and  from  the  filtrate  an  amount 
is  measured  off  corresponding  to  10  c.c.  of  the  original  urine. 

Execution  of  the  Titration.  Nearly  the  whole  quantity  of  the  mer- 
curic-nitrate solution,  which  is  judged  from  the  specific  gravity  of  the  urine 
to  be  the  minimum  amount  required,  is  added  at  once,  and  immediately 
aftei-wards  the  quantity  of  soda  solution  necessary,  as  indicated  by  the 
table.  If  the  mixture  becomes  yellowish  in  color,  then  too  much  mercury 
solution  has  been  added  and  another  determination  must  be  made.  If  the 
test  remains  white,  and  if  a  drop  taken  out  and  placed  on  a  glass  plate 
with  a  dark  background  and  stirred  with  a  drop  of  a  thin  paste  of  sodium 
bicarbonate  does  not  give  a  yellow  color,  the  addition  of  mercury  solution  is 
continued  by  adding  repeatedly  at  first  J  and  later  tV  c.c,  and  testing 
after  each  addition  in  the  following  way :  A  drop  of  the  mixture  is  placed 
on  a  glass  plate  with  a  dark  background  beside  a  small  drop  of  the  bicar- 
bonate paste.  If  the  color  after  stirring  the  two  drops  together  is  still 
white  after  a  few  seconds,  then  more  mercury-  solution  must  be  added;  if, 
on  the  contrarj%  it  is  yellowish,  then — if  not  too  much  mercurj'  solution 
has  been  added  by  inattention — the  result  to  xV  c.c.  has  been  found.  B}^ 
this  approximate  determinatio,  which  is  sufficient  in  many  cases,  we  haven 


560  URINE. 

fixed  the  minimum  amount  of  mercury  solution  necessary  to  add  to 
the  quantity  of  urine  in  question,  and  we  now  proceed  to  the  final  deter- 
mination. 

A  second  quantity  of  the  filtrate,  corresponding  to  10  c.c.  of  the 
original  urine,  is  filtered,  and  the. same  quantity  of  mercury  solution 
added  at  one  time  as  was  found  necessary  to  produce  the  final  reaction, 
and  immediately  after  the  corresponding  amount  of  soda  solution,  which 
must  not  indicate  the  end  of  the  reaction.  Then  continue  adding  the 
mercury  solution  xV  <'-c.  at  a  time  without  neutralizing  with  soda,  until 
a  drop  taken  out  and  mixed  with  the  soda  solution  gives  a  yellow  color- 
ation. If  this  final  reaction  is  obtained  after  the  addition  of  0.1-0.2  c.c, 
then  the  titration  may  be  considered  as  finished.  If,  on  the  contrary,  a 
larger  quantity  is  necessary,  the  addition  of  the  mercury  solution  must 
be  continued  until  a  final  reaction  is  obtained  with  simple  carbonate,  and 
the  titration  repeated  again,  adding  the  quantity  of  mercury  solution  used 
in  the  previous  test  at  one  time,  and  also  adding  the  corresponding  amount 
of  soda  solution.  If  then  the  end  reaction  is  obtained  by  the  addition  of 
i^Tj  c.c,  the  titration  may  be  considered  as  finished. 

If  in  each  titration  a  quantity  of  filtrate  containing  urine  and  baryta 
correspcnding  to  10  c.c.  of  the  original  urine  is  used,  then  the  calculations 
are  ver\'  simple,  since  1  c.c.  of  mercuric-nitrate  solution  corresponds  to 
0.01  gram  of  urea.  As  the  mercuiy  solution  is  made  for  a  2  per  cent  urea 
solution,  and  as  the  filtrate  of  urine  and  liaryta  generally  contains  less 
urea  (if  the  quantity  of  urea  is  above  2  per  cent,  it  is  easy  to  avoid  any  mis- 
take by  diluting  the  urine  at  the  Ijeginning  of  the  operation),  a  mistake 
occurs  here  which  can  be  corrected  in  the  following  way,  according  to 
Pfluger:  To  the  measured  volume  of  the  filtrate  from  the  urine  (the 
filtrate  with  bars'ta  after  neutralization  with  nitric  acid,  precipitation  vnth 
silver  nitrate  and  filtration)  the  quantity  of  normal  soda  solution  employed 
is  added,  and  from  this  sum  the  volume  of  mercury  solution  used  is  sub- 
tracted. The  remainder  is  then  multiplied  by  0.08,  and  the  product  sub- 
tracted from  the  number  of  cubic  centimeters  of  mercury  solution  used. 
For  example,  if  the  filtrate  (urine  and  baryta  + nitric  acid  +  silver  nitrate) 
measured  25.8  c.c,  and  the  number  of  cubic  centimeters  of  soda  solution 
used  in  the  titration  was  13.8  c.c,  and  of  the  mercury  solution  20.5  c.c,  we 
have  then  20.5-|(39.6-20.5)X0.081  =  20.5-  1.53=  18.97,  and  the  corrected 
quantity  of  mercur\^  solution  is  therefore  18.97  c.c.  If  the  cubic  centi- 
meters of  the  filtrate  (in  this  case  25.8  c.c.)  correspond  to  10  c.c.  of  the 
original  urine,  then  the  amount  of  urea  is  18.97X0.01  =  0.1897=18.97  p.  m. 
urea. 

Besides  the  urea  other  nitrogenous  constituents  of  the  urine  are  precipi- 
tated by  the  mercury  solution.  In  the  titration  we  really  do  not  obtain 
the  quantity  of  urea,  but,  as  PFLtJGER  has  shown,  the  total  quantity  of 


ESTIMATION  OF  UREA.  561 

nitrogen  in  the  urine  expressed  as  urea.  As  urea  contains  46.67  per  cent 
N,  the  total  quantity  of  nitrogen  in  the  urine  may  be  calculated  from  the 
quantity  of  urea  found.  The  results  obtained  by  this  calculation  corre- 
spond well,  according  to  Pfluger,  with  the  results  found  for  the  total 
nitrogen  as  determined  by  Kjeldahl's  method. 

Glassivl\nx  ^  has  recently  suggested  a  modification  of  the  Liebig- 
PFLiJGER  titration  method  which  consists  in  precipitating  the  urea  with 
an  excess  of  mercuric-nitrate  solution  and  then  determining  the  excess 
of  mercuric  nitrate  in  the  filtrate  by  means  of  ammonium  sulphocyanide. 

Among  the  methods  suggested  for  the  special  estimation  of  urea,  that  of 
Morner-Sjoqvist,  in  combination  with  Folix's  method,  is  perhaps  the 
most  tiTistv.orthy  and  readily  performed.  For  this  reason  only  this  method 
will  be  given  in  detail,  while  we  must  refer  to  sj^ecial  works  for  the  other 
methods,  such  as  Buxsex's  method  with  its  many  modifications  as  sug- 
gested by  Pfluger,  Bohlaxd  and  Bleibtreu.^ 

Principle  oj  M orner-Sjoqvist' s  Method.^  According  to  this  method  the 
nitrogenous  constituents  of  the  urine,  with  the  exception  of  urea,  ammonia, 
hippuric  acid,  creatinine,  and  traces  of  allantoin,  are  precipitated  by  a  mix- 
ture of  alcohol  and  ether  after  the  addition  of  a  solution  of  bariun  chloride 
and  barium  hydrate  or  in  the  presence  of  sugar  with  solid  barium  hydiate. 
The  urea  is  determined  in  the  concentrated  filtrate,  after  dri\-ing  off  the 
ammonia,  by  Kjeldahl's  nitrogen  estimation.  Because  of  the  slight  error 
due  to  the  presence  of  liippuric  acid  and  creatinine,  several  modifications 
have  been  suggested  by  Salaskix  and  Zaleski  and  by  Brauxsteix  * 
These  errors  are  best  prevented,  according  to  ^Iorxer,  by  the  use  of 
FoLix's  method. 

Principle  of  Folin's  Method.^  On  heating  urea  with  hydrochloric  acid 
and  cr}'stalUne  magnesium  chloride,  which  melts  in  its  water  of  cr}-stalUza- 
tion  at  112-115°  C.  and  then  boils  at  about  150-155°  C,  the  urea  is  com- 
pletel}"  decomposed,  while  no  appreciable  decomposition  of  the  hippuric 
acid  and  creatinine  takes  place.  The  ammonia  produced  from  the  urea  is 
distilled  off  and  determined  by  titration.  The  amount  of  ammonia  pre- 
viously existing  in  the  urine  must  be  specially  determined. 

Determination  of  Urea  hy  the  Morner-Sfoqvist  and  Folin  Method.^  Five 
c.c.  of  the  urine  are  treated  with  1.5  grams  of  powdered  barium  hydroxide, 
and  when  as  much  of  this  is  dissob-ed  as  possilde  by  gentlv  mixing,  it  is 

»Ber.  d.  d.  chem.  Gesellsch.,  39. 
2  Pfliiger's  Arch.,  38,  43,  and  44. 

^Skand.  Arch.  f.  Physiol.,  2,  and  Monier,  ibid.,  14,  where  the  recent  literature 
may  also  be  found. 

*  Braunstein,  Zeitschr.  f.  physiol.  Chem.,  31;  Salaskin  and  Zaleski,  ibid.,  28. 

5/&;(/.,32,  36,  and37. 

'  See  Morner,  Skand.  Arch.  f.  Physiol.,  14. 


562  URINE. 

precipitated  by  100  c.c.  of  the  alcohol  and  ether  mixture  (^  vol.  ether).  On 
the  iollowing  day  it  is  filtered  and  the  precipitate  washed  with  the  alcohol 
and  ether  mixture.  The  alcohol  and  ether  are  distilled  off  from  the  filtrate 
at  about  55°  C.  (not  above  60°  C).  The  remaining  hquid  is  treated  with 
2  c.c.  of  hydrochloric  acid  of  sp.  gr.  1.124  (for  5  c.c.  urine),  and  carefully 
transferred  to  a  flask  of  200  c.c.  capacit}',  and  evaporated  to  dryness  on 
the  water-ljath.  Then  add  20  grams  of  cr\'staUine  magnesium  chloride  to 
the  contents  of  the  flask  and  2  c.c.  of  concentrated  hydrochloric  acid,  and 
boil  on  a  wire  gauze  over  a  small  flame  for  two  hours,  making  use  of  a  proper 
return  cooler.  After  cooUng  it  is  diluted  to  about  |  to  1  liter  with  water,  the 
ammonia  completely  distilled  off  after  making  it  alkaline  with  caustic  soda, 
and  the  ammonia  collected  in  standard  acid.  After  boiling  in  order  to  drive 
off  the  CO 2  and  cooling,  the  acid  is  retitrated.  Corrections  must  be  made  for 
the  ammonia  of  the  urine  and  for  that  contained  in  the  magnesium  chloride. 
If  a  special  determination  of  the  preformed  ammonia  has  been  made, 
then  a  direct  treatment  of  the  urine  according  to  Folin  (nevertheless  after 
the  evaporation  of  the  urine  with  hydrochloric  acid)  gives  good  results. 
In  the  presence  of  sugar  the  treatment  of  the  urine  with  barium  hydroxide 
is  absolutely  necessary^  according  to  ]\1orxer,  othenvise  the  humin  sub- 
stances produced  from  the  sugar  take  up  and  retain  nitrogen. 

Knop-Hufner's  method  ^  is  based  on  the  fact  that  urea,  by  the  action  of 
sodium  hypobromite,  spHts  into  water,  carbon  dioxide  (which  dissolves  in  the 
alkali),  and  nitrogen,  whose  volume  is  measured  (see  page  555).  This  method 
is  less  accurate  than  the  preceding  ones,  and  therefore  in  scientific  work  it  is 
discarded.  It  is  of  value  to  the  physician  and  for  practical  j^urposes,  because 
of  the  ease  and  rapidity  ■nith  which  it  may  be  performed,  even  though  it  may 
not  give  very  accurate  results.  For  practical  piu-poses  a  number  of  different 
apparatuses  have  been  constructed  to  facilitate  the  use  of  this  method. 

For  the  quantitative  estimation  of  urea  in  blood  or  other  animal  fluids, 
as  well  as  in  the  tissues,  Schoxdorff  has  proposed  a  method  where  the 
proteins  and  extractives  are  first  precipitated  by  a  mixture  of  phospho- 
tungstic  acid  and  hydrochloric  acid,  and  then  the  filtrate  made  alkahne 
with  lime.  The  quantity  of  ammonia  formed  on  heating  a  part  of  this 
filtrate  to  150°  C.  with  phosphoric  acid  and  the  amount  of  carbon  diox- 
ide produced  by  heating  the  other  part  to  150°  C.  are  determined.  In 
regard  to  the  principles  of  this  method,  as  well  as  to  the  details,  we  refer 
to  the  original  article  (PFi.iJGER's  Arch.,  62).  See  also  Hoppe-Seyler- 
Thierfelder's  Handbuch,  7.  Aufl. 

Urein  is  the  name  given  by  Ovid  Moor  to  a  product  which  he  obtained  by 
extracting  urine,  which  had  been  evaporated  to  a  syrujD,  with  absolute  alcohol 
and  precipitating  the  urea  with  alcohol  containing  oxalic  acid,  or  by  cooling  and 
treatment  with  alcohol.  Urein  is  a  golden-yellow  oil  which  is  poisonous;  it 
reduces  permanganate  in  the  cold,  and  it  forms  the  chief  portion  of  the  nitro- 
genous extractives  of  urine.  There  is  no  doubt  but  that  urein  is  a  mixture  of  sub- 
stances.     According  to  MoOR,^  the  amount  of  urea  in  the  urine  is  only  about 

'  Knop,  Zeitschr.  f.  analj^l.  Chem.,  9;  Hiifncr,  Journ.  f.  prakt.  Cheni.  (N.  F.),  3. 
In  regard  to  the  extensive  literature,  see  Huppert-Neubauer,  10.  Aufl.,  304,  and  follow- 
ing. 

2  O.  Moor,  Bull.  Acad,  de  St.  Petersbourg,  14  (also  Maly's  Jahresber.,  31,  415), 
and  Zeitschr.  f.  Biologie,  44  and  45,  and  Zeitschr.  f.  physiol.  Chem.,  40. 


CREATININE.  563 

one-half  that  ordinarily  given,  and  he  has  suggested  a  new  method  for  the  deter- 
mination of  the  true  quantity  of  urea.  The  possibility  that  in  the  urine  we  have 
other  bodies  besides  urea  which  have  been  determined  mth  the  urea  cannot 
be  denied  a  'priori.  From  the  investigations  published  so  far  it  must  be  said 
that  Moor's  assertions  are  not  sufficiently  groimded.' 

\H 
Carbamic   Acid,  CH3N02=CO  <qjj-.     This  acid  is  not  known  in  the  free  state, 

but  only  as  salts.  Ammonium  carbamate  is  produced  by  the  action  of  dry  ammo- 
nia on  dry  carbon  dioxide.  Carbamic  acid  is  also  produced  by  the  action  of 
potassium  permanganate  on  protein  and  several  other  nitrogenous  organic  bodies. 

The  occcurrence  of  carbamic  acid  in  human  and  animal  urines  has  already 
been  considered  in  connection  with  the  formation  of  urea.  The  calciiun  salt, 
which  is  soluble  in  water  and  ammonia  but  insoluble  in  alcohol,  is  the  most 
important  in  the  detection  of  this  acid.  The  solution  of  the  calcium  salt  in  water 
becomes  cloudy  on  standing,  but  much  more  quickly  on  boiling,  and  calcium  car- 
bonate separates.  Nolf,  MACLEonand  Haskixs  have  made  experiments  as  to  the 
method  of  formation  of  carbamic  acid.  The  latter  have  indicated  a  new  method 
for  the  quantitative  estimation  of  carbamates.^ 

Carbamic-acid  ethylester  (urethane),  as  shown  by  Jaffe,^  maj'  pass,  by  the 
mutual  action  of  alcohol  and  urea,  into  the  alcoholic  extract  of  urine  when  one 
is  working  ^v-ith  large  quantities. 

.NH CO 

Creatinine,  C4H7X3O,  or  NH : q/  I       ,  is  generally  considered  as 

\X(CH3).CH2 

the  anhydride  of  creatine  (see  page  455)  found  in  the  muscles.  It  occurs 
in  human  urine  and  in  that  of  certain  mammaUa.  It  has  also  been  found 
in  ox-blood,  milk,  though  in  veiy  small  amounts,  and  in  the  flesh  of  certain 
fishes. 

.loHNSOx's  statement  that  the  creatinine  of  the  urine  is  different  from  that 
produced  by  the  action  of  acids  on  creatine  is  incorrect  according  to  Toppelius  and 

POMMEREHNE,    WOERNER   and   ThELEN.^ 

The  quantity  of  creatinine  in  human  urine  is,  in  a  grown  man  voiding  a 
normal  quantity  of  urine  in  the  course  of  a  da}-,  0.6-1.3  grams  (Xeubauer), 
or  on  an  average  1  gram.  Johnson  5  found  1.7-2.1  grams  per  day,  and 
similar  results  have  been  obtained  by  Hoogexhuyze  and  Verploegh.^ 
The  quantity  of  creatinine  vsith  a  diet  free  from  meat  is,  according  to 
FoLEN'.'^  somewhat  variable  for  different  individuals,  but  is  constant  for 

'See    Kubiabko,  Maly's  Jahresber.,  31,  41.5;    Erben,  Zeitschr.  f.  physiol.  Chem. 
38;    Folin,  ibid.,  37;    Gies,  Journ.  Amer.  Chem.  See,  25;    Haskins,  Anier.  Journ.  of 
Physiol.,  12;    Lippich,  Zeitschr.  f.  phy,siol.  Chem.,  48. 

^  Nolf ,  Zeitschr.  f.  physiol.  Chem.,  23;  Macleod  and  Haskins,  Amer.  Journ.  of 
Physiol.,  12. 

^Zeitschr.  f.  physiol.  Chem.,  14. 

*S.  Johnson,  Proceed.  Roy.  Soc,  42,  43;  Chem.  News,  55;  Toppelius  and  Pern- 
merehne,  Arch.  f.  Phann.,  234;  Woemer,  Arch.  f.  {Axia.i.  u.)  Physiol.,  1898. 

*  Huppert-Neubauer,  Hamanalyse,  10.  Aufl.,  387. 

*  Zeitschr.  f.  physiol.  Chem.,  4(5. 

'  Amer.  Journ.  of  Physiol.  13;   af.  Klercker,  Hofmeister's  Beitrage,  8. 


564  URINE. 

the  same  person.  He  found  the  quantity  never  below  1  gram  and  often 
between  1.3  and  1.7  grams.  Nurslings  also  eliminate  creatinine,  although 
the  quantity  is  only  small  (Hoogenhuyze  and  Verploegh).  The  quantity 
of  creatinine  is  dependent  upon  the  food  in  so  far  as  it  is  increased  l)y  meat 
diet,  but  otherwise,  according  to  Folin,  it  is  not  dependent  upon  the  food. 
The  creatinine  is,  according  to  him,  the  product  of  the  endogenous  metabo- 
lism of  the  cells,  and  its  quantity  does  not  dejDend,  as  was  also  shown  later 
by  Klercker,  upon  the  quantity  of  protein  food  introduced  and  catabo- 
lizcd.  The  eUmination  of  creatinine,  therefore,  does  not  run  parallel  with 
the  elimination  of  urea  and  is  not  correspondingly  greater  with  food  very 
rich  in  protein  than  with  food  very  poor  in  protein. 

The  statements  as  to  the  behavior  of  the  creatinine  elimination  with 
work  are  very  contradictory .^  Hoogenhuyze  and  Verploegh,  who  made 
use  of  a  much  more  trustworthy  method  of  quantitative  estimation  than 
their  predecessors,  find  that  muscular  activity  as  a  rule  does  not  cause 
any  rise  in  the  creatinine  elimination,  and  that  in  man  such  a  rise  with 
work  occurs  only  when  the  body  is  obliged  to  live  upon  its  own  tissues. 
Little  is  known  about  the  behavior  of  creatinine  in  diseases.  In  cases 
with  an  increase  in  metabolism  the  quantity  is  said  to  rise,  while  in  other 
cases,  as  in  ansemia  and  cachexia  mth  reduced  metabolism,  the  quantity 
is  lessened. 

Creatinine  crystalhzes  in  colorless,  shining  monoclinic  prisms  which 
differ  from  creatine  crystals  in  not  becoming  white  with  loss  of  water  when 
heated  to  100°  C.  It  dissolves  in  11  parts  cold  water,  but  more  easily  in 
warm  water.  It  is  difficultly  soluble  in  cold  alcohol,  but  the  statements 
in  regard  to  its  solubilities  differ  widely.-  It  is  more  soluble  in  warm  alcohol 
and  nearly  insoluble  in  ether.  In  alkaline  solution  creatinine  is  converted 
into  creatine  ver}^  easily  on  warming. 

Creatinine  gives  an  easily  soluble  crystalline  compound  with  hydro- 
chloric acid.  A  solution  of  creatinine  acidified  with  mineral  acids  gives 
cr}'stalline  precipitates  with  phosphotungstic  and  phosphomolybdic  acids 
even  in  very  dilute  solutions  (1:10  000)  (Kerner,  Hofmeister^).  It  is 
precipitated,  like  urea,  by  mercuric-nitrate  solution  and  also  by  mercuric 
chloride.  On  treating  a  dilute  creatinine  solution  with  sodium  acetate  and 
then  with  mercuric  chloride  a  precipitate  of  glassy  globules  having  the 
composition  4 (C4H7N30.HCl.HgO)3HgCl2  separates  on  standing  some  time 
(Johnson).  Among  the  compounds  of  creatinine,  that  with  zinc  chloride, 
creatinine  zinc  chloride,  (C4H7N30)2ZnCl2,  is  of  special  interest.     This  com- 


*  The  literature  on  this  subject  may  be  found  in  Hoogenhuyze  and  Verploegh,  I.  c. 
^  See   Huppert-Neubauer,   10.  Aufi.     and    Hoppe-Seyler-Thierfolder's   Handbuch, 
Aufl. 
^  Kerner,  Pfliiger's  Arch.,  2;  Hofmeister,  Zeitschr.  f.  physiol.  Chem.,  5. 


CREATININE.  565 

bination  is  obtained  when  a  sufficiently  concentrated  solution  of  creatinine 
in  alcohol  is  treated  with  a  concentrated,  faintly  acid  solution  of  zinc 
chloride.  Free  mineral  acids  dissolve  the  compound,  hence  they  must 
not  be  present;  this,  however,  may  be  prevented  by  an  addition  of  sodium 
acetate.  In  the  impure  state,  as  ordinarily  obtained  from  urine,  creati- 
nine zinc  chloride  forms  a  sandy,  yellowish  powder  which  under  the  micro- 
scope appears  as  fine  needles  forming  concentric  groups,  mostly  complete 
rosettes  or  yellow  balls  or  tufts,  or  grouped  as  brushes.  On  slowly  crys- 
tallizing or  when  very  pure,  more  sharply  defined  prismatic  crystals  are 
obtained.     The  compound  is  sparingly  soluble  in  water. 

Creatinine  acts  as  a  reducing  agent.  JMercuric  oxide  is  reduced  to- 
metallic  mercurj^,  and  oxaHc  acid  and  methylguanidine  (methyluramine) 
are  formed.  Creatinine  also  reduces  cupric  hydrate  in  alkaline  solution^ 
forming  a  colorless  soluble  compound,  and  only  after  continued  boiling 
with  an  excess  of  copper  salt  is  free  subxoide  of  copper  formed.  Creatinine 
interferes  with  Trommer's  test  for  sugar,  partly  because  it  has  a  reducing 
action  and  partly  by  retaining  the  copper  suboxide  in  solution.  The  com- 
pound -with  copper  suboxide  is  not  soluble  in  a  saturated  soda  solution, 
and  if  a  Uttle  creatinine  is  dissolved  in  a  cold  saturated  soda  solution  and 
then  a  few  drops  of  Fehling's  reagent  added  a  white  flocculent  compound 
separates  after  heating  to  50-60°  C.  and  then  cooling  (v.  ^Iaschke's  ^ 
reaction).  An  alkahne  bismuth  solution  (see  Sugar  Tests)  is  not  reduced 
by  creatinine. 

If  we  add  a  few  drops  of  a  freshly  prepared  verj'  dilute  sodium-nitro- 
prusside  solution  (sp.  gr.  1.003)  to  a  dilute  creatinine  solution  (or  to  the 
urine)  and  then  a  few  drops  of  caustic  soda,  a  ruby-red  liquid  is  obtained 
which  quickly  turns  yellow  again  (Weyl's  ^  reaction) .  If  the  cold  yellow 
solution  is  neutralized  and  treated  with  an  excess  of  acetic  acid  a  crystalline 
precipitate  of  a  nitroso-compound  (C4H6N4O2)  of  creatinine  separates  on 
stirring  (Kramm  3).  If,  on  the  contrary,  the  yellow  solution  is  treated  with 
an  excess  of  acetic  acid  and  heated,  the  solution  becomes  first  green  and  then 
blue  (Salkowski  ^) ;  finally  a  precipitate  of  Prussian  blue  is  obtained.  If 
a  solution  of  creatinine  in  water  (or  urine)  is  treated  with  a  watery  solution 
of  picric  acid  and  a  few  drops  of  a  dilute  caustic-soda  solution,  a  red  colora- 
tion lasting  several  hours  occurs  immediately  at  the  ordinarj^  temperature, 
which  turns  yellow  on  the  addition  of  acid  (Jaffe's  ^  reaction).  Acetone 
gives  a  more  reddish-yellow  color.  Dextrose  gives  with  this  reagent  a  red 
coloration  only  after  heating. 

*  Zeitschr.  f.  analyt.  Chem.,  17. 

^  Ber.  d.  deut.sch.  chem.  Gesellsch.,  11, 
'  Centralbl.  f.  d.  med.  Wissensch.,  1897. 

*  Zeitschr.  f.  physiol.  Chem.,  4. 
Uhid.,  10. 


566  URINE. 

In  preparing  creatinine  from  urine  the  creatinine  zinc  chloride  is  first 
prepared  according  to  Neubauer's  ^  method.  One  hter  or  more  of  urine  is 
treated  with  milk  of  lime  until  alkaline  and  then  CaCl2  solution  is  added 
until  all  the  phosphoric  acid  is  precipitated.  The  filtrate  is  evaporated  to  a 
syrap  after  faintly  acidifying  with  acetic  acid  and  this  is  treated  while  still 
warm  with  97  per  cent  alcohol  (about  200  c.c.  for  each  liter  of  urine).  After 
■about  twelve  hours  it  is  filtered  and  the  filtrate  treated  first  with  a  little 
.sodium  acetate  and  then  ^^■ith  an  acid-free  zinc  chloride  solution  of  a  specific 
gravity  of  1.20  (aliout  2  c.c.  for  each  liter  of  urine.)  After  thorough  stir- 
ring it  is  allowed  to  stand  forty -eight  hours  and  the  precipitate  is  collected 
on  a  filter  and  washed  with  alcohol.  The  creatinine  zinc  chloride  is  dis- 
solved in  hot  water,  boiled  with  lead  oxide,  filtered,  the  filtrate  decolorized 
by  animal  charcoal,  evaporated  to  dryness,  and  the  residue  extracted  with 
strong  alcohol  (which  leaves  the  creatinine  undissolved) .  The  alcoholic  ex- 
tract is  evaporated  to  the  point  of  crystallization,  and  the  crystals  purified 
by  recrj^stallization  from  water. 

Creatinine  may  also  be  prepared  from  urine  by  precipitating  with  a 
mercuric-chloride  solution  according  to  either  Maly's  or  Johnson's  ^ 
process. 

The  best  method  for  preparing  creatinine  is  the  following,  suggested  by 
FoLiN.^  The  creatinine  is  first  precipitated  as  the  double  picrate  of  creati- 
nine and  potassium  by  means  of  picric  acid  according  to  Jaffe's  method, 
and  then  this  precipitate,  while  still  moist,  is  decomposed  by  KHCO3  and 
water.  The  solution,  which  contains  the  creatinine  besides  potassium 
carbonate  and  small  amounts  of  impurities,  is  neutralized  with  sulphuric 
acid  and  the  sulphate  precipitated  by  alcohol.  The  creatinine  is  now  con- 
verted into  the  doul)le  zinc-chloricle  salt  and  this  last  treated  with  moist 
lead  hydroxide.  Alter  the  removal  of  the  lead  the  solution  contains  a  mix- 
ture of  f-reatinine  and  creatine,  which  last  is  completely  transformed  into 
creatinine  by  heating  tor  48  hours  with  normal  sulphuric  acid.  After  exact 
neutralization  with  barium-hydrate  solution  it  is  concentrated  to  the  point 
of  crystallization. 

The  quantitative  eMiwation  of  creatinine  may  be  performed  according  to 
Neubauer's  method  for  the  preparation  of  creatinine,  or  more  simply  ]iy 
Salkowski's  4  modification  01  this  method.  240  c.c.  of  the  urine  freed  from 
])roteid  (Ijy  l^oiling  with  acid)  and  from  sugar  (l\y  lermentation  with  yeast) 
are  made  alkaline  with  milk  of  lime,  and  precipitated  by  CaClo  and  made  up 
to  300  c.c:  250  c.c.  (=200  c.c.  urine)  of  this  are  measured  off,  neutralized 
or  made  only  faintly  acid  with  acetic  acid  and  evaporated  to  aboiit  20  c.c, 
then  thoroughly  stirred  with  an  equal  volume  of  al^solute  alcohol,  and 
completely  transferred  to  a  100,  c.c.  flask  which  contains  some  alcohol, 
the  residue  in  the  dish  being  washed  with  alcohol.  On  thorough  shaking 
and  cooling,  the  flask  is  filled  up  to  the  100-c.c.  mark  with  al)solute  alcohol 
and  allowed  to  stand  twenty-four  hours.  80  c.c.  (=160  c.c.  urine)  of 
the  filtrate  are  collected  in  a  beaker  and  treated  with  0.5-1  c.c.  of  zinc- 
chloride  solution,  and  the  covered  beaker  is  left  standing  in  a  cool  place 

*  Ann.  d.  Chom.  u.  Pharm.,  119. 

^Maly,  Annal.  d.  Chem.  u.  Pharm,,  159;  Johnson,  Proceed.  Roy.  See,  43. 
^  Zeitschr.  f.  physiol.  Chem.,  41. 

*  Zeitschr.  f.  physiol.  Chem.,  10  and  14. 


CREATININE   AND   XANTHOCREATININE.  567 

for  two  or  three  daj-s.  The  precipitate  is  collected  on  a  small  dried  and 
weighed  filter,  using  the  filtrate  to  wash  the  crystals  Irom  the  beaker. 
After  allowino-  the  crystals  to  completely  drain  off,  they  are  washed  with 
a  little  alcohol  until  the  filtrate  gives  no  reaction  for  chlorine,  and  dried  at 
100°  C.  100  parts  of  creatinine  zinc  chloride  contain  62.44  parts  of  creati- 
nine. As  the  precipitate  is  never  quite  pure,  the  quantity  of  zinc  must  be 
carefully  determined,  in  exact  experiments,  by  evaporating  with  nitric  acid, 
heating,  washing  the  oxide  of  zinc  with  water  (to  remove  any  NaCl),  drj'ing, 
heating,  and  weighing.  22.4  parts  zinc  oxide  correspond  to  100  parts 
creatinine  zinc-chloride.  Instead  of  weighing,  the  nitrogen  can  be  deter- 
mined by  Kjeldahl's  method  and  the  creatinine  calculated  from  this. 

FoLiN  1  has  suggested  a  colorimetric  method  for  determining  creatinine 
which  is  based  upon  Jaffe's  picric-acid  reaction  and  is  as  follows:  10  c.c. 
of  the  urine  are  treated  in  a  graduated  flask  of  500  c.c.  capacity  with  15  c.c. 
of  a  1.2  per  cent  solution  of  picric  acid  and  5  c.c.  of  a  10  per  cent  NaOH 
solution.  After  shaking  and  allowing  to  stand  for  5  minutes  it  is  diluted 
with  water  to  500  c.c.  and  mixed.  This  solution  is  now  compared  in  a 
DuBOSCQ  colorimeter  with  a  ^  normal  potassium-bichromate  solution. 
The  latter  solution  has  in  a  layer  8  mm.  thick  exactly  the  same  intensity 
of  color  as  a  layer  8.1  mm.  thick  of  a  solution  of  10  milligrams  creatinine 
after  the  addition  of  15  c.c.  picric-acid  solution  and  5  c.c.  NaOH  solution 
and  dilution  to  500  c.c.  The  calculations  are  simjile.  For  example,  in 
case  the  urine  tested  in  a  layer  7.2  mm.  thick  has  the  same  color  as  the 
dichromate  solution  in  a  layer  8  mm.  thick,  then  the  quantitv  of  creatinine 

8  1 
in  10  c.c.  of  the  urine  will  be  =;7^X  10,  or  11.25  milligrams.      This  method 

is  not  only  simple,  but  also,  according  to  Folin,  Hoogenhuyze  and  Ver- 
PLOEGH,  gives  much  more  trustworthy  results  than  Neubauer's  method. 
In  regard  to  other  methods,  see  the  works  of  Kolisch  and  Gregor.^ 

Xanthocreatinine,  CgHjoN^O.  This  body,  which  was  first  prepared  from  meat 
extract  by  Gautier,  has  been  found  by  Monari  in  dog's  urine  after  the  injection 
of  creatinine  into  the  abdominal  cavity,  and  in  human  urine  after  several  hours 
of  exhausting  marching.  According  to  Colasanti  it  occurs  to  a  relatively  greater 
extent  in  lion's  urine.  Stadthagex  ^  considers  the  xanthocreatinine  isolated 
from  human  urine  after  strenuous  muscular  activity  as  impure  creatinine. 

Xanthocreatinine  forms  thin  sulphur-yellow  plates,  similar  to  cholesterin, 
which  have  a  bitter  taste.  It  dissolves  in  cold  water  and  in  alcohol,  and  gives 
a  crystalline  compound  with  hydrochloric  acid  and  a  double  compound  with 
gold  and  platinum  chloride.  It  gives  a  compound  with  zinc  chloricle,  which 
crystallizes  in  fhie  needles.     Xanthocreatinine  has  a  poisonous  action. 

HN— CO 

Uric  Acid,  Ur,  C5H4N4O3,  2,  6,  8-trioxypurine  =  OC     C— NH\ 

I      II  >C0,  has 

HN— C— NH/ 


^  Z:^itschi.  f.  phyiol.  Chc-ni.,  41. 

2  Kolisch,  Centralbl.  f.  innere  Med.,  1895;   Gregor,  Zeitschr.  f.  physiol.  Chem.,  31. 

3  Gautier,  Bull,  de  racad.  de  med.  (2)  15,  and  Bull,  de  la  .-oc.  chira.  (2),  48:  Monari. 
Maly's  Jahresber.,  17;  Colasanti,  Arch.  ital.  d.  Biologie,  15,  Fasc.  3;  Stadthagen, 
Zeitschr.  f.  klin.  Med.,  15. 


568  URINE. 

been  prepared  synthetically  by  Horbaczewski  by  fusing  urea  and  glycocoll 
or  by  heating  trichlorlactic-acid  amide  with  an  excess  of  urea.  Behrend 
and  RoosEN  prepared  it  from  isodialuric  acid  and  urea;  it  is  also  readily 
produced  from  isouric  acid  on  boiling  with  hydrochloric  acid  (E.  Fischer 
and  Tullner),  and  finally  E.  Fischer  and  Ach  ^  have  prepared  uric  acid 
from  pseudouric  acid  by  heating  with  oxalic  acid  to  145°  C. 

On  strongly  heating  uric  acid  it  decomposes  with  the  formation  of 
urea,  hydrocyanic  acid,  cyanuric  acid,  and  ammonia.  On  heating  with 
concentrated  hydrochloric  acid  in  sealed  tubes  to  170°  C.  it  splits  into 
glycocoll,  carbon  dioxide,  and  ammonia.  By  the  action  of  oxidizing  agents 
splitting  and  oxidation  take  place,  and  either  monoureides  or  diureides 
are  produced.  By  oxidation  with  lead  peroxide,  carbon  dioxide,  oxalic 
acid,  urea,  and  allantoin,  which  last  is  glyoxyldiureide,  are  produced  (see 
below) .  By  oxidation  with  nitric  acid  in  the  cold,  urea  and  a  monoureide, 
the  mesoxalyl  urea,  or  alloxan,  are  obtained,  C5H4N403  +  0  +  H20= 
C4H2N2O4+  (NH2)2CO.  On  warming  with  nitric  acid,  alloxan  yields 
carbon  dioxide  and  oxalyl  urea,  or  parabanic  acid,  C3H2N2O3.  By  the 
addition  of  water  the  parabanic  acid  passes  into  oxaluric  acid,  C3H4N2O4, 
traces  of  which  are  found  in  the  urine  and  which  easily  splits  into  oxalic 
acid  and  urea.  In  alkaline  solution  uric  acid  may,  by  taking  up  water 
and  oxygen,  be  transformed  into  a  new  acid,  uroxanic  acid,  C5H8N4O6, 
which  may  then  be  changed  into  oxonic  acid,  C4H5N3O4.2  Uric  acid  may, 
as  F.  and  L.  Sestini  as  well  as  Gerard  have  shown,  undergo  bacterial 
fermentation  with  the  formation  of  urea.  According  to  Ulpiani  and 
CiNGOLANi,^  uric  acid  is  quantitatively  split  hereby  into  urea  and  carbon 
dioxide,  according  to  the  equation 

C5H4N403  +  2H20  +  30  =  3C02+2CO(NH2)2. 

Uric  acid  occurs  most  abundantly  in  the  urine  of  birds  and  of  scaly 
ampliibians,  in  which  animals  the  greater  part  of  the  nitrogen  of  the  urine 
appears  in  this  form.  Uric  acid  occurs  frequently  in  the  urine  of  carniv- 
orous mammalia,  but  is  sometimes  absent;  in  urine  of  herl^ivora  it  is  habitu- 
ally present,  though  only  as  traces;  in  human  urine  it  occurs  in  greater 
but  still  small  and  variable  amounts.  Traces  of  uric  acid  are  also  found 
in  several  organs  and  tissues,  as  in  the  spleen,  lungs,  heart,  pancreas,  liver 
(especially  in  Ijirds),  and  in  the  brain.  It  haljitually  occurs  in  the  blood 
of  birds.     Traces  have  been  found  in  human  l^lood  under  normal  con- 

"  Horbaczewski,  Monatshefte  f.  Chem.,  6  and  S;  Behrend  and  Roosen,  Ber.  d.  d. 
chem.  Gesellsch.,  21;   Fischer  and  Tiillner,  ibid.,  35;   Fischer  and  Ach,  ibid.,  28. 

^  See  Sundwik,  Zeitschr.  f.  physiol.  Chem.,  20  and  41;  also  Behrend,  Annal.  d.  Chem. 
u.  Pharm.,  333. 

'  See  Chem.  Centralbl.,  1903,  where  the  other  investigators  are  cited,  and  Centralbl. 
f.  Physiol.,  19. 


URIC  ACID.  569 

ditions.  Under  pathological  conditions  it  occurs  to  an  increased  extent 
in  the  blood,  as  in  pneumonia  and  nephritis,  but  especially  in  leuca-mia  and 
sometimes  also  in  arthritis.  Uric  acid  also  occurs  in  large  quantities  in 
''chalk-stones,"  certain  urinary  calcuU,  and  in  guano.  It  has  also  been 
detected  in  the  urine  of  insects  and  certain  snails,  as  also  in  the  wings  (which 
it  colors  white)  of  certain  butterflies  (Hopkins).^ 

The  amount  of  uric  acid  eliminated  with  human  urine  is  subject  to 
considerable  individual  variation,  but  amounts  on  an  average  to  0.7  gram 
per  day  on  a  mixed  diet.  The  ratio  of  uric  acid  to  urea  varies  considerably 
with  a  mixed  diet,  but  is  on  an  average  1:50-1:70.  In  new-born  infants 
and  in  the  first  days  of  life  the  elimination  of  uric  acid  is  relatively  in- 
creased, and  the  relation  between  uric  acid  and  urea  has  been  found  to 
be  1:6.42-17.1. 

We  used  to  ascribe  an  increasing  action  upon  the  elimination  of  uric 
acid  to  protein  food,  l)ut  the  investigations  of  Hirschfeld,  Rosenfeld  and 
Orgler,  Sivex,  Burian  and  Schur,^  and  many  others  have  positively 
proved  that  a  diet  rich  in  protein  does  not  itself  increase  the  elimination 
of  uric  acid,  but  only  according  to  the  amount  of  nucleins  or  purine  bodies 
contained  therein.  The  common  statement  that  the  elimination  of  uric 
acid  is  smaller  with  a  vegetaljle  diet  than  with  an  animal  diet,  when  the 
quantity  may  be  2  grams  or  more  per  twenty-four  hours,  is  explained  by 
this.3 

The  statements  in  regard  to  the  influence  of  other  circumstances,  as 
also  of  different  substances,  on  the  elimination  of  uric  acid  are  rather  con- 
tradictory. This  is  in  part  due  to  the  fact  that  the  older  investigators 
used  an  inaccurate  method  (Heintz),  and  also  that  the  extent  of  uric-acid 
elimination  is  dependent  in  the  first  place  upon  the  individuality.  Thus 
the  statements  in  regard  to  the  action  of  drinking-water  *  and  of  alkalies  ^ 
are  very  contradictory.  Certain  medicines,  such  as  quinine  and  atropine, 
diminish,  while  others,  such  as  pilocarpine  and  also,  as  it  seems,  salicyhc 
acid, 6  increase  the  elimination  of  uric  acid. 

'  Philos.  Trans.  Roy.  Soc,  186,  B,  661. 

2  See  the  extensive  review  of  the  literature  in  Wiener,  "Die  Hamsaure,"  in  Ergeb- 
nisse  der  Physiologie,  1,  Abt.  1,  1902. 

^  J.  Ranke,  Beobachtungen  und  Versuche  i'lber  die  Ausscheidung  der  Hamsaure, 
etc.  (Miinchen,  1858);  Mares,  Centralbl.  f.  d.  med.  Wissensch.,  1888;  Horbaczewski, 
Wien.  Sitzungsber.,  100,  Abt.  3,  1891.  In  regard  to  the  action  of  various  diets  the 
reader  is  referred  to  the  above-cited  authors,  and  especially  to  A.  Hermann,  .\rch.  f. 
klin.  Med.,  43,  and  Camerer,  Zeitschr.  f.  Biologic,  33,  and  Folin,  Amer.  Journ.  of 
Physiol.,  13. 

*  See  Schondorff,  Pfliiger's  Arch.,  46,  which  contains  the  pertinent  literature. 

^  See  Clar,  Centralbl.  f.  d.  med.  Wissen&ch.,  1888;  Haig,  Journ.  of  Physiol.,  8;  and 
A.  Hermann,  Arch.  f.  klin.  Med.,  43. 

"  See  Bohland,  cited  from  Maly's  Jahresber.,  26;   Sclireiber  and  Zaudy,  ibid.,  30. 


570  URINE. 

Little  is  known  with  positi\eness  in  regard  to  the  elimination  of  uric 
acid  in  disease.  In  acute  diseases  with  crises  the  eUmination  of  uric  acid  is 
increased  after  the  crisis,  wliile  the  older  statements  that  the  uric  acid  is 
habitually  increased  in  fevers  has  been  contradicted  by  many.  The  state- 
ments in  regard  to  the  elimination  of  uric  acid  in  gout  and  nephritis  are  also 
uncertain  and  contradictory.  In  leucaemia  the  ehmination  is  increased 
absolutely  as  well  as  relatively  to  the  urea,  and  the  relationship  between 
the  uric  acid  and  urea  (total  nitrogen  recalculated  as  urea)  may  be  even 
1:9,  while  under  normal  conditions,  according  to  different  investigators, 
it  is  1:10  to  66  to  100. ^ 

Formation  of  Uric  Acid  in  the  Organism.  Since  Horbaczewski  first 
showed  that  uric  acid  could  be  produced  by  oxidation  from  the  nuclein-rich 
spleen-pulp  or  nucleins  outside  of  the  body,  he  also  showed  that  nucleins 
when  introduced  into  the  animal  body  caused  an  increase  in  the  elimination 
of  uric  acid.  These  observations  have  been  confirmed,  and  at  the  same 
time  developed  by  the  work  of  a  great  number  of  investigators,  and  we 
are  sure  that  uric  acid  can  Ije  produced  from  purine  bodies  either  outside 
or  inside  the  animal  body,  and  also  that  food  rich  in  nucleins  (especially 
the  thymus  gland)  increases  the  ehmination  of  uric  acid  and  purine  bases 
(alloxuric  bases  ^).  The  original  view  of  Horbaczewski,  that  the  nucleins 
do  not  directly  cause  an  increased  elimination  of  uric  acid,  but  indirectly 
by  causing  a  leucocytosis  with  a  conse  uent  destruction  of  leucocytes,  has 
been  nearly  generally  discarded.  A.t  present  it  is  considered  that  a  direct 
formation  of  uric  acid  from  the  nucleins  takes  place  by  the  transformation 
of  the  purine  bases  of  the  nucleins  into  uric  acid. 

The  uric  acid,  in  so  far  as  it  is  produced  from  nuclein  bases,  is  in  part 
deri\ed  trom  the  nucleins  of  the  destroyed  cells  of  the  body  and  in  part 
irom  the  nucleins  or  free  purine  bases  introduced  with  the  food.  It  is  there 
fore  possible  to  admit  with  Burian  and  Schur  ^  of  a  double  origin  for  the 
uric  acid  as  well  as  the  urinar}'  purines  (all  ]:)urine  bodies  ot  the  urine,  in- 
cluding the  uric  acid),  namely,  an  endogenous  and  an  exogenous  origin. 
Burian  and  Schur  attempted  to  determine  the  quantity  of  endogenou.s 
urinary  purines  by  feeding  with  sufficient  food,  but  as  free  as  possil^le  from 
purine  bodies,  and  they  found  that  tliis  quantity  was  constant  for  every 
indi\idual,  while  it  was  variable  for  different  persons.  The  observations 
of  SivEX,  RocKWooD,*  and  others  have  also  led  to  the  same  results.     Other 

'  In  regard  to  the  extensive  literature  on  the  elimination  of  uric  acid  in  disease 
we  must  refer  to  special  works  on  internal  diseases. 

^  As  it  is  not  within  the  scope  of  this  book  to  enter  into  a  discussion  of  the  numer- 
ous researches  on  this  subject,  we  will  refer  to  Wiener,  "Die  Harnsaure,"  Ergebnisse 
der  Physiol.,  1,  Abt.  1,  1902. 

5  PHuger's  Arch.,  80,  87,  and  94. 

*  Amer.  Joum.  of  Physiol.,  12. 


FORMATION   OF    URIC  ACID.  571 

investigators,  such  as  Sciireiber  and  "Waldvogel,  Loewi,  and  Folix,^ 
have  arrived  at  somewhat  different  results,  or  they  draw  differentde  ductions 
from  their  observations;  still  this  does  not  change  the  essential  fact,  that 
the  uric  acid  originating  from  the  nucleins  is  partly  endogenous  and  partly 
exogenous,  and  that  the  amount  of  endogenous  uric  acid  is  onh'  ver}-  slightly 
dependent  upon  the  protein  content  of  the  food. 

In  man  and  other  mammalia  the  greatest  amount  if  not  all  of  the  uric 
acid  originates  from  the  nucleins  or  their  purine  bases.  This  formation  of 
uric  acid  seems  to  be  of  an  enzymotic  kind.  After  it  was  shown  that  certain 
organs,  such  as  the  liver  and  spleen,  had  the  power  of  converting  oxypurines 
into  uric  acid  in  the  presence  of  oxygen  (Horbaczew^ski,  Spitzer  and 
Wiener  2),  recently  Schittexhelm,  Buriax,  Jones  and  Partridge,^  by 
more  careful  investigations  have  shown  that  enzymes  of  different  lands 
act  together.  By  means  of  the  two  deamidizing  enzymes  adenase  and 
guanase  the  adenine  and  guanine  are  transformed  into  hypoxanthine 
and  xanthine  respectively,  and  from  the  latter  by  means  of  an  oxidizing 
enzyme,  called  xanthine  oxidase  b}^  Burian,  the  uric  acid  is  formed.  The 
deamidizing  enzymes  seem  to  be  present  in  most  organs,  yet  there  exists, 
in  tliis  regard,  a  marked  difference  between  certain  animals;  thus  guanase 
occurs  in  the  ox-spleen  but  not  in  the  pig-spleen  (Jones  and  Winterintz)  . 
The  oxidase  occurs  especially  in  the  spleen  (though  not  in  the  dog  spleen, 
according  to  Schittex'^helm)  and  liver,  but  also  in  the  muscles  and  lungs. 
Still,  as  ScHiTTEXHELM  4  has  especially  shown,  a  verj-  marked  difference 
exists  in  animals,  and  the  activity  of  the  organs  of  various  animals  requires 
a  very  thorough  investigation. 

Jones  and  Austrl^n  ^  have  carried  on  investigations  on  the  occurrence 
in  different  organs  of  pigs,  dogs,  and  rabbits,  of  enzymes  taking  part  in  the 
nuclein  metabolism.  The  occurrence  of  these  enz^^mes  in  the  liver  is  of 
special  interest.  In  the  above-mentioned  animals  and  in  the  ox  they  found 
the  following:  The  l^eef-liver  contains  guanase,  adenase,  and  xanthine 
oxidase,  and  produces  uric  acid  from  guanine  as  well  as  from  adenine. 
Guanase  is  absent  from  the  pig-liver,  while  adenase  and  xanthine  oxidase 
are  present.  In  these  animals  the  liver  forms  uric  acid  from  adenine  but 
not  from  guanine.  The  rabbit-liver  does  not  contain  any  adenase  and 
hence  uric  acid  is  formed  only  from  guanase.  wliile  the  dog-liver,  on  the 
contrar}',  which  contains  guanase  but  neither  adenase  nor  xanthine  oxidase, 
cannot  form  uric  acid  from  <ruanine  nor  from  adenine. 

'  Schreiber  and  Waldvogel,  .\rch.  f.  exp.  Path.  u.  Phann.,  42;  O.  Loewi,  ibid.,  44 
and  4o:    Folin,  Amer.  Journ.  of  Physiol.,  13. 

'  See  foot-note  2,  page  .570. 

^  Schittenhelm,  Zeitschr.  f.  physiol.  Chem.,  42,  43,  45,  and  46;  Burian,  ibid.,  43- 
Jones  and  Partridge,  ibid..  42;   .Jones  and  Wintemitz  ibid.,  44;  Jones,  ibid.,  45. 

*  Zeitschr.  f.  physiol.  Chem.,  46. 

'  Zeitschr.  f.  physiol.  Chem.,  48. 


572  URINE. 

In  birds  the  conditions  are  different,  v.  Mach  ^  has  shown  that  in  these 
animals  a  part  of  the  uric  acid  may  be  formed  from  the  purine  bodies.  The 
ehief  quantity  of  uric  acid,  however,  is  undoubtedly  formed  in  birds  by 
synthesis. 

The  formation  of  uric  acid  in  birds  is  increased  by  the  administration 
of  ammonium  salts  (v.  Schroder),  and  urea  acts  in  a  similar  manner  in 
these  animals  (^Ieyer  and  Jaffe).  Minkowski  observed  in  geese  with 
extirpated  livers  a  ver\'  significant  decrease  in  the  elimination  of  uric  acid, 
while  the  elimination  of  ammonia  was  increased  to  a  corresponding  degree. 
This  indicates  a  participation  of  ammonia  in  the  formation  of  uric  acid  in 
the  organism  of  birds ;  and  as  ^Iinkowski  has  also  found  after  the  extirpa- 
tion of  the  liver  that  considerable  amounts  of  lactic  acid  occur  in  the  urine, 
it  is  probable  that  the  uric  acid  in  birds  is  produced  in  the  liver  by  syn- 
thesis, perhaps  from  lactic  acid  and  ammonia;  although,  as  Salaskin  and 
Zaleski  and  Lang  have  shown,  after  the  extirpation  of  the  liver  primarily 
an  increase  in  the  formation  of  lactic  acid  occurs  and  this  causes  an  in- 
crease in  the  eUmination  of  ammonia  (neutralization  ammonia) .  The  direct 
proof  for  the  uric-acid  formation  from  ammonia  and  lactic  acid  in  the 
hver  of  birds  has  been  given  by  Kow'alewski  and  Salaskin  ^  by  means 
of  blood-transfusion  experiments  on  geese  with  extirpated  livers.  They 
observed  a  relatively  abundant  formation  of  uric  acid  after  the  addition 
of  ammonium  lactate  and  a  still  greater  formation  after  arginine.  They  not 
only  consider  ammonium  lactate  but  also  amino-acids  as  substances  from 
which  the  uric  acid  can  be  produced  in  the  liver  by  synthesis.  Of  these 
leucine,  glycocoU,  and  aspartic  acid  increase  the  elimination  of  uric  acid  in 
birds  (v.  Knieriem^),  but  whether  they  are  first  decomposed  with  the 
splitting  off  of  ammonia  is  still  unknown. 

The  possibilit}'  of  a  formation  of  uric  acid  from  lactic  acid  has  been 
shown  in  another  manner  by  Wiener,*  namely,  by  feeding  birds  with  urea 
and  lactic  acid  and  different  non-nitrogenous  substances,  oxy-,  keto-,  and 
dibasic  acids  of  the  aUphatic  series.  The  dibasic  acids,  with  a  chain  of  3 
carbon  atoms  or  their  ureides,  showed  themselves  most  activ^e  as  uric-acid 
formers,  and  Wiener  is  therefore  of  the  opinion  that  the  active  substances 
must  first  be  converted  in'o  dibasic  acids.  By  the  attachment  of  a  urea 
residue  the  corresponding  ureide  is  produced,  according  to  Wiener,  and 
from  this  the  uric  acid  is  derived  by  the  attachment  of  a  second  urea  residue. 

'  Arch.  f.  exp    Path.  u.  Pharm.,  24. 

*  V.  Schroder,  Zeitschr.  f.  physiol.  Chem.,  2;  Meyer  and  Jaff^,  Ber.  d.  d.  Chem. 
Gesellsch.,  10;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  21  and  31;  Salaskin  and 
Zaleski,  Zeitschr.  f.  physiol.  Chem.,  29;  Lang,  'ibid.,  32;  Kowalewski  and  Salaskin, 
ibid.,  33. 

'  Zeitschr.  f.  Biologie.  13. 

*  Hofmeister's  Beitrage,  2.  See  also  Arch.  f.  exp.  Path.  u.  Pharm.,  42,  and  Ergeb- 
nisse  d.  Physiol.,  1,  Abt.  1,  1902. 


FORMATION    OF  URIC   ACID.  573 

Among  the  substances  tested,  only  tartronic  acid  and  its  ureide,  dialuric  acid, 
have  shown  themselves  active  in  the  experiments  with  the  isolated  organs,  and 
Wiener  therefore  also  considers  that  the  other  acids  must  be  first  converted  into 
tartronic  acid  by  oxidation  or  reduction.  From  lactic  acid,  CH3.CH(0H).C00H, 
we  first  obtain  tartronic  acid,  C00H.CH(0H).C00H,  which  by  the  attachment 

of  a  urea  residue  forms  dialuric  acid,  CO<ytt pQ>CHOH,  and  from  this,  by  the 

attachment  of  a  second  urea  residue,  uric  acid  is  formed. 

We  cannot  give  any  positiv^e  answer  as  to  the  question  w^hether  uric  acid 
is  formed  by  synthesis  also  in  man  and  other  mammalia.  Wiener  has 
in  part  reported  experiments  which  seem  to  indicate  a  synthetic  uric-acid 
formation  in  the  isolated  mammalian  liver,  and  he  has  also  obtained  an 
increase  in  the  uric-acid  elimination,  although  only  a  slight  one,  after  feed- 
ing lactic  acid  and  dialuric  acid  to  man.  According  to  Burian  ^  we  have 
for  the  present  no  proof  of  a  synthetical  formation  of  uric  acid  in  the 
mammalian  liver.  Dialuric  acid  and  tartronic  acid,  according  to  him,  do 
not  cause  any  marked  uric-acid  formation  with  extracts  of  the  ox-liver  in 
the  absence  of  purine  bases;  on  the  contraiy  they  accelerate  the  enzymotic 
oxidation  of  purine  bases  and  hence,  according  to  Burian,  this  explains, 
perhaps,  the  increase,  in  uric-acid  elimination. 

The  liver  seems  to  be  the  organ  in  birds  where  the  synthetical  formation 
of  uric  acid  occurs,  and  the  fact  that  it  was  possible  for  ^Minkowski  ^  to 
arrest  the  uric-acid  formation  by  the  extirpation  of  the  liver  apparently 
shows  that  the  liver  is  the  only  organ  taking  part  in  this  synthesis.  If  a 
synthesis  of  uric  acid  also  occurs  in  man  and  other  mammalia  we  must  con- 
sider the  liver  as  at  least  one  of  the  organs  taking  part  in  the  work,  as 
shown  by  W^iener's  investigations.  The  liver,  spleen,  and  muscles  are 
considered  as  the  most  important  organs  for  the  oxidative  uric-acid  forma- 
tion from  nucleins  and  purine  bases,  but  it  must  not  be  forgotten  that  these 
organs  in  various  animals  have  a  somewhat  different  behavior  in  this  re- 
gard. 

Uric  acid  w^hen  introduced  into  the  mammalian  organism  is,  as  first 
shown  by  Wohler  and  Frerichs  for  the  dog  and  later  substantiated  by 
several  experimenters,^  in  great  part  destroyed  and  more  or  less  com- 
pletely changed  into  urea.  This  does  not  seem  to  be  the  same  for  all  ani- 
mals. In  rabbits,  according  to  Wiener,  the  uric  acid  is  destroyed  with 
the  formation  of  glycocoU  as  an  intermediate  step.  The  statements  are 
very  contradictory  in  regard  to  carnivora.  According  to  an  older  view, 
which  has  received  support  by  the  recent  investigations  of  Salkoavski.  a 
part  of  the  uric  acid  introduced  into  dogs  is  eliminated  as  allantoin,  which 

•  Zeitschr.  f.  physiol.  Chem.,  43. 
2  1.  c. 

'Wohler  and  Frerichs,  Annal.  d.  Chem.  u.  Pharm.,  Go.  See  also  Wiener,  Ergeb- 
nisse  der  Physiologie,  1,  Abt.  1. 


571  URINE. 

is  also  true  according  to  Mendel  and  Brown  for  cats.  The  correctness 
of  tliis  \iew  is  denied  by  Wiener,  Pohl  and  Poduschka,  but  the  recent 
investigations  of  ^Mendel  and  White  i  give  further  proofs  of  its  correct- 
ness. The  possibihty  of  a  uric-acid  destiTiction  in  man,  with  allantoin  as 
an  intermediary  step,  cannot,  for  the  same  reasons,  l)e  denied. 

The  demoUtion  of  uric  acid  seems  to  be  possible,  according  to  the  nu- 
merous researches  of  Chassevant  and  Richet,  Ascoli,  Jacoby,  W^iener, 
Schittenhelm,  Burian,  Almagia  and  Pfeiffer,^  in  several  organs,  such 
as  the  liver,  kidneys,  muscle,  and  bone-marrow,  although  its  behavior 
differs  in  various  animals. 

From  this  power  of  the  various  organs  of  destroying  uric  acid  it  follows 
that  the  quantity  of  uric  acid  eliminated  is  not  a  sure  indication  of  the 
amount  of  the  acid  formed.  We  must  admit,  therefore,  that  a  part  of  the 
uric  acid  formed  in  the  body  is  destroyed  in  a  similar  manner  to  that  intro- 
duced from  without.  Burian  and  Schur  ^  have  indeed  suggested  a  factor, 
the  so-called  "integral  factor,"  with  which  the  quantity  of  uric  acid  elimi- 
nated in  the  twenty-four  hours  must  be  multiplied  in  order  to  find  the 
quantitv  of  uric  acid  formed  during  this  time.  According  to  them,  car- 
nivora  eliminate  unchanged  about  -gV-  3V  of  the  uric  acid  introduced  into 
the  circulation,  rabbits  about  \,  and  man  4. 

Properties  and  Reactions  of  Uric  Acid.  Pure  uric  acid  is  a  white,  odor- 
less, and  tasteless  powder  consisting  of  very^  small  rhombic  prisms  or  plates. 
Impure  urio  acid  is  easily  obtained  as  somewhat  larger,  colored  crystals. 

In  rapid  crystallization,  small,  thin,  four-sided,  apparently  colorless, 
rhombic  prisms  are  formed,  which  can  be  seen  only  by  the  aid  of  the  micro- 
scope, and  these  sometimes  appear  as  spools  because  of  the  rounding  of 
their  obtuse  angles.  The  plates  are  sometimes  six-sided,  irregularly  devel- 
oped; in  other  cases  they  are  rectangular  with  partly  straight  and  partly 
jagged  sides;  and  in  other  cases  they  show  still  more  irregular  forms,  the 
so-called  dumb-bells,  etc.  In  slow  cr\''stallization,  as  when  the  urine  de- 
posits a  sediment  or  when  treated  with  acid,  large,  invariably  colored  cr\'S- 
tals  separate.  Examined  with  the  microscope  these  crystals  always  appear 
vellow  or  yellowish  brow^n  in  color.  The  most  common  type  is  the  whet- 
stone shape,  formed  by  the  rounding  off  of  the  obtuse  angles  of  the  rhoml)ic 

•Wiener,  Arch.  f.  exp.  Path.  ii.  Pharm.,  40  and  42,  and  Ergebnisse  der  Physiologie, 
1  Abt.  1;  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.,  48;  Poduschka,  ib-id.,  44;  Salkowski, 
Zeitschr.  f.  physiol.  Chem.,  35,  and  Ber.  d.  d.  Chem.  Gesellsch.,  9;  Mendel  and  Brown, 
Amer.  Journ.  of  Physiol,  3;   Mendel  and  White,  Jbid.,  12. 

2  Chassevant  et  Richet,  Compt.  rend.  Soc.  biolog.,  49;  Ascoli,  Pfliiger's  Arch.,  72; 
Jacoby,  Virchow's  Arch.,  157;  Wiener,  Arch.  f.  exp.  Path.  u.  Pharm.,  42,  and  Centralbl. 
f.  Physiol.,  18;  Schittenhelm,  Zeitschr.  f.  physiol.  Chem.,  43  and  45;  Burian,  ibid., 
43;   Almagia,  Hofmeister's  Beitrage,  7;   Pfeiffer,  ibid.,  7. 

s  Pfliiger's  Arch.,  87. 


PROPERTIES  OF  URIC  ACID.  575 

plate.  The  whetstones  are  generally  connected  together,  two  or  more 
crossing  each  other.  Besides  these  forms,  rosettes  of  prismatic  crj'stals, 
irregular  crosses,  brown-colored  rough  masses  of  broken-up  crystals  and 
prisms  occur,  as  well  as  other  forms. 

Uric  acid  is  insoluble  in  alcohol  and  ether;  it  is  rather  easily  soluble  in 
boiling  glycerine,  but  very  insoluble  in  cold  water,  in  39  480  parts  at 
18°  C.  (His  and  Paul).  At  this  temperature,  according  to  them,  9.5  per 
cent  of  the  uric  acid  is  dissociated  in  the  saturated  solution.  Because  of 
the  reduction  in  the  dissociation  on  the  addition  of  strong  acids  uric  acid 
is  soluble  with  difficulty  in  the  presence  of  mineral  acids.  It  is  soluble 
in  a  warm  solution  of  sodium  diphosphate,  and  in  the  presence  of  an  excess 
of  uric  acid,  monophosphate  and  acid  urate  are  produced.  It  is  ordinarily 
assumed  that  sodium  diphosphate  forms  a  solvent  for  the  uric  acid  in  the 
urine,  but  according  to  Smale  the  monophosphate  has  only  a  slight  sol- 
vent action.  According  to  RiJDEL  ^  urea  is  an  imjwrtant  solvent,  but  this 
statement  has  not  been  confirmed  by  the  observations  of  His  and  Paul. 
Uric  acid  is  not  only  dissolved  by  alkalies  and  alkali  carbonates,  but  also  by 
several  organic  bases,  such  as  ethylamine  and  propylamine,  piperidine  and 
piperazine.  Uric  acid  dissolves  without  decomposing  in  concentrated 
sulphuric  acid.  It  is  completely  precipitated  from  the  urine  by  picric 
acid  (Jaffe^).  XJric  acid  gives  a  chocolate-brown  precipitate  with  phos- 
photungstic  acid  in  the  presence  of  hydrochloric  acid. 

Uric  acid  is  dibasic  and  correspondingly  forms  two  series  of  salts,  neu- 
tral and  acid.  Of  the  alkali  urates  the  neutral  potassium  and  lithium  salts 
are  most  easily  soluble  and  the  ammonium  salt  dissolves  with  difficultv. 
The  acid  alkali  urates  are  very^  insoluble  and  separate  as  a  sediment  (sedi- 
mentum  loteritium)  from  concentrated  urine  on  cooling.  The  salts  with 
alkaline  earths  are  very  insoluble. 

If  a  little  uric  acid  in  substance  is  treated  on  a  porcelain  dish  with  a 
few  drops  of  nitric  acid,  the  uric  acid  dissolves  on  warming  with  a  strong 
development  of  gas,  and  after  thoroughly  drying  on  the  water-bath  a 
beautiful  red  residue  is  obtained,  which  turns  a  purjole-red  (ammonium 
purjuirate  or  murexide)  on  the  addition  of  a  little  ammonia.  If,  instead  of 
the  ammonia,  we  add  a  little  caustic  soda  (after  cooling),  the  coior  becomes 
deeper  blue  or  1:)luish  violet.  This  color  disappears  quickly  on  warminff, 
differing  from  certain  xanthine  bodies.  This  reaction  is  called  the  murexide 
test. 

If  uric  acid  is  converted  into  alloxan  by  the  careful  action  of  nitric  acid 
and  the  excess  of  acid  carefully  expelled,  on  treating  this  with  a  few  droj)s 


'  His,  Jr.,  and  Paul,  Zeitschr.  f.  physiol.  Chem.,  31;    Smale,  Centralbl.  f.  Physiol., 
9;  Riidel,  Arch.  f.  exp.  Path.  u.  Phann.,  50. 
2  Zeitschr.  f.  physiol.  Chem.,  10. 


576  UIUNE. 

of  concentrated  sulphuric  acid  and  commercial  benzene  (containing  thio- 
phene)  a  beautiful  blue  coloration  is  obtained  (Dt^nk;es'  reaction  i). 

Uric  acid  does  not  reduce  an  alkaline  solution  of  bismuth,  while,  on  the 
contrary,  it  reduces  an  alkaline  cupric-hydrate  solution.  In  the  presence  of 
only  a  little  copper  salt  we  obtain  a  white  precipitate  consisting  of  cuprous 
urate.  In  the  presence  of  more  copper  salt  red  cuprous  oxide  separates. 
The  compound  of  uric  acid  with  cuprous  oxide  is  formed  when  copper 
salts  are  reduced  by  dextrose  or  a  bisulphite  in  alkaline  solution  in  the 
presence  of  a  sufficient  amount  of  urate. 

If  a  solution  of  uric  acid  in  water  containing  alkali  carbonate  is  treated 
with  magnesium  mixture  and  then  a  silver-nitrate  solution  added,  a  gelati- 
nous precipitate  of  silver-magnesium  urate  is  formed.  If  a  drop  of  uric  acid 
dissolved  in  sodium  carbonate  is  placed  on  a  piece  of  filter-paper  which  has 
been  previously  treated  with  silver-nitrate  solution,  a  reduction  of  silver 
oxide  occurs,  producing  a  brownish-black  or,  in  the  presence  of  only  0.002 
milligram  of  uric  acid,  a  yellow  spot  (Schiff's  test). 

The  precipitation  of  free  uric  acid  from  its  alkaU  salts  by  means  of 
acids  can  be  prevented  to  some  extent  by  the  presence  of  thymic  acid  or 
nucleic  acid  (Goto  2),  It  is  questionable  whether  this  is  of  any  physio- 
logical importance. 

Preparation  of  Uric  Acid  from  Urine.  Filtered  normal  urine  is  treated 
with  20-30  c.c.  of  25  per  cent  hydrochloric  acid  for  each  liter  of  urine. 
After  forty -eight  hours  collect  the  crystals  and  purify  them  by  redissolving 
in  dilute  alkali,  decolorizing  with  animal  charcoal  and  re]3recipitating  with 
hydrochloric  acid.  Large  quantities  of  uric  acid  are  easily  obtained  from 
the  excrements  of  serpents  by  boiling  them  with  dilute  caustic  potash  (5  per 
cent)  until  no  more  ammonia  is  developed.  A  current  of  carbon  dioxide 
is  passed  through  the  filtrate  until  it  barely  has  an  alkaline  reaction;  dis- 
solve the  separated  and  washed  acid  potassium  urate  in  caustic  potash,  and 
precipitate  the  uric  acid  in  the  filtrate  by  addition  of  an  excess  of  hydro- 
chloric acid. 

Quantitative  Estimation  of  Uric  Acid  in  the  Urine.  As  the  older  method 
suggested  by  Heintz,  even  after  recent  modifications,  gives  inaccurate 
results,  it  will  not  l)e  considered  here. 

Salkowski  and  Ludwig's^  method  consists  in  precipitating  by  silver 
nitrate  the  uric  acid  from  the  urine  previously  treated  with  magnesium 
mixture,  and  weighing  the  uric  acid  obtained  from  the  silver  precipitate. 
Uric  acid  determinations  by  this  method  are  often  performed  according  to 
the  suggestion  of  E.  Ludwig,  which  requires  tne  following  solutions: 


'  Journ.  de  Pharm.  et  de  Chim.,  18.     Cited  from  Maly's  Jahresber.,  18. 

2  Zeitschr.  f.  pliysiol.  Chem.,  30. 

^  Salkowski,  Virchow's  Arch.,  52;  Pfliiger's  Arch.,  5;  Salkowski,  Laboratory  Manual 
of  Physiol,  and  Path.  Chem.,  translated  by  Orndorff,  1904;  Ludwig,  Wien.  med. 
Jahrbuch,  1884,  and  Zeitschr.  f.  anal.  Chem.,  24. 


ESTIMATION   OF  URIC  ACID.  577 

1.  An  AMMONiACAL  siLVER-xiTRATE  SOLUTION,  which  Contains  in  1  liter  26 
grams  of  silver  nitrate  and  a  quantity  of  ammonia  sufficient  to  redissolve  com- 
pletely the  precipitate  produced  by  the  first  addition  of  ammonia.  2.  Magne- 
sia MiXTLRE.  Dissolve  100  grams  <if  crystallized  magnesium  chloride  in  water, 
add  ammonia  until  the  liquid  smells  strongly  of  it,  and  enough  ammonium 
chloride  to  dissolve  the  precipitate;  then  dilute  the  solution  to  1  liter.  3.  Sodium- 
sulphide  SOLUTION.  Dissolv  elO  grams  of  caustic  soda  which  is  free  from  nitric 
acid  and  nitrous  acid  in  1  liter  of  water.  One  half  of  this  solution  is  completely 
saturated  with  sulphuretted  hych"ogen  and  then  mixed  with  the  other  half. 

The  concentration  of  the  three  solutions  is  so  arranged  that  10  c.c.  of 
each  is  sufficient  for  100  c.c.  of  the  urine. 

100-200  c.c,  according  to  concentration,  of  the  filtered  urine  freed 
from  protein  (by  boiling  after  the  addition  of  a  few  drops  of  acetic  acid)  is 
poured  into  a  beaker.  In  anotlier  vessel  mix  10-20  c.c.  of  the  silver  solu- 
tion with  10-20  c.c.  of  the  magnesia  mixture  and  add  ammonia,  and  when 
necessars'  also  some  ammonium  chloride,  until  the  mixture  is  clear.  This 
solution  is  added  to  the  urine  wliile  stirring,  and  the  mixture  allowed  to 
stand  quietly  for  half  an  hour.  The  precipitate  is  collected  on  a  filter, 
waslied  with  ammoniacal  water,  and  then  returned  to  the  same  beaker  by 
the  aid  of  a  glass  rod  and  a  wash-bottle,  without  destroying  the  filter. 
NoW'  heat  to  boiling  10-20  c.c.  of  the  alkali-sulphide  solution,  which  has 
previously  been  diluted  with  an  equal  volume  of  water,  and  allow  this  solu- 
tion to  flow  through  the  above  filter  into  the  beaker  containing  the  silver 
precipitate;  wash  with  boiling  water,  and  warm  the  contents  of  the  beaker 
on  a  water-bath  for  a  time,  stirring  constantly.  After  cooling,  filter  into  a 
porcelain  dish,  wash  the  filter  with  boiling  water,  acidify  the  filtrate  with 
hydrochloric  acid,  evaporate  it  to  about  15  c.c,  add  a  few  drops  more  of 
hydrochloric  acid,  and  allow  it  to  stand  for  twenty-four  hours.  The  uric 
acid  which  has  cr\'stallized  is  collected  on  a  small  weighed  filter,  washed 
wdth  w^ater,  alcohol,  ether,  and  carbon  clisulphide,  dried  at  100-110°  C,  and 
w^eighed.  For  each  10  c.c  of  aqueous  filtrate  we  must  add  0.00048  gram 
uric  acid  to  the  quantity  found  directly.  Instead  of  the  weighed  filter- 
paper  a  glass  tube  filled  with  glass  wool  as  described  in  other  handbooks 
may  be  substituted  (Ludw^g).  Too  intense  or  too  long  continued  heat- 
ing with  the  alkali  sulphide  must  be  prevented,  otherwise  a  part  of  the 
uric  acid  may  be  decomposed. 

Salkowski  deviates  from  this  procedure  by  precipitating  the  urine  first 
with  a  magnesium  mixture  (50  c.c.  to  200  c.c.  urine),  filling  up  to  300  c.c, 
and  filtering.  Of  the  filtrate,  200  c.c.  is  precipitated  by  10-15  c.c.  of  a 
3  per  cent  silver-nitrate  solution.  The  silver  precipitate  is  shaken  with  200- 
300  c  c  of  water  acidified  with  a  few  drops  of  hydrochloric  acid,  decomposed 
by  sulphuretted  hydrogen,  heated  to  boiling,  the  silver-sulphide  precipitate 
boiled  with  fresh  water,  filtered,  the  filtrate  concentratecl  to  a  few  cubic 
centimetres,  treated  with  5-8  drops  of  hydrochloric  acid,  and  allowed  to 
stand  until  the  next  day. 

Hopkins'  method  is  based  on  the  fact  that  the  uric  acid  is  completely 
precipitated  from  the  urine  as  ammonium  urate  on  saturating  with  am 
monium  chloride.  The  uric  acid  can  either  be  weighed  after  being  set  free 
by  hydrochloric  acid  or  it  can  be  determined  in  several  ways — by  titration 
with  potassium  permanganate  or  by  the  Kjeldahl  method.  Several  modi- 
fications of  this  method  have  been  worked  out  by  Folin,  Folin  and  Schaf- 


578  URINE. 

FEE,  WoRNER,  and  JoLLES.i  The  last  named  converts  the  uric  acid 
into  urea  by  oxidation  with  potassium  permanganate  in  sulphuric-acid 
solution  and  then  determines  the  quantity  of  this  by  sodium  hypobromite. 
Of  these  methods  we  shall  descril^e  only  that  suggested  by  Folin-Schaffer. 

Folin-Schaffer  Method.  Treat  300  c.c.  urine  with  75  c.c.  of  a  solution 
containing  500  grams  of  ammonium  sulphate,  5  grams  of  uranium  acetate, 
and  60  c.c.  of  10  per  cent  acetic  acid  in  a  liter,  and  filter  after  five  minutes. 
This  removes  an  unknown  constituent  of  the  urine  (a  protein  substance) 
which  would  otherwise  contaminate  the  uric  acid.  Take  125  c.c.  of  the  fil- 
trate (corresponding  to  100  c.c.  of  the  vu'ine)  and  add  5  c.c.  of  concentrated 
ammonia.  After  twenty -four  hours  the  precipitate  is  filtered  off  and  washed 
free  from  chlorine  on  the  filter  by  means  of  an  ammonium-sulphate  solution. 
The  precipitate  is  washed  off  the  filter  by  water  (total  100  c.c.)  into  a  flask, 
treated  with  15  c.c.  of  concentrated  sulphuric  acid,  and  titrated  at  60-63° 
C.  with  N/20  potassium-permanganate  solution.  Each  cubic  centimeter 
of  this  solution  corresponds  to  3.75  milligrams  uric  acid.  Because  of  the 
solubility  of  the  ammonium  urate  a  correction  of  3  milUgrams  must  be 
added  for  exevy  100  c.c.  of  the  urine. 

In  regard  to  the  numerous  other  methods  for  estimating  uric  acid,  we 
must  refer  to  special  w^orks  on  the  subject,  and  especially  to  Huppert- 
Neubauer . 

Purine  Bases  (Alloxuric  Bases).  The  alloxuric  bases  (purine  bases) 
found  in  human  urine  are  xanthine,  guanine,  hypoxanthine,  adenine,  yara- 
xanthine,  heteroxanthine,  episarkine,  epiguanine,  1-methylxanthine,  and  car- 
nine.  The  occurrence  of  guanine  and  camine  (Pouchet)  is,  according  to 
Kruger  and  Salomon ,2  not  positive Ij^  shown.  The  quantity  of  these 
bodies  in  the  urine  is  extremely  small  and  varies  in  different  individuals. 
Flatow  and  Reitzexstein  ^  found  15.6-45.1  milligrams  in  the  urine  voided 
during  twenty -four  hours.  The  quantity  of  alloxuric  bases  in  the  urine  is 
increased  regularly  after  feeding  with  nucleus  nucleins  or  food  rich  in  nu- 
cleins,  and  after  free  destruction  of  leucocytes.  The  quantity  is  especially 
increased  in  leucsemia.  We  have  a  number  of  observations  on  the  elimina- 
tion of  these  bodies  in  different  diseases,  l)ut  they  are  hardly  trustworthy  on 
account  of  the  inaccuracy  of  the  methods  used  in  the  determinations.  It 
must  also  be  remarked  that  the  three  alloxuric  bases,  heteroxanthine,  para- 
xanthine,  and  1-methylxanthine,  which  form  the  chief  mass  of  the  alloxuric 
bases  of  the  urine,  are  derived,  according  to  the  investigations  of  Albanese, 
BoNDZYNSKi  and  Gottlieb,  E.  Fischer,  ]\I.  Kriiger  and  G.  Salomon,  and 


•Hopkins,  Journ.  of  Path,  and  Bact.,  1893,  and  Proceed.  Roy.  Soc,  52;  Folin, 
Zeitsehr.  f.  physiol.  Chem.,  24;  Folin  and  Schaflfer,  ibid.,  32;  Worner,  ibid.,  29;  Jolles, 
ibid.,  29,  and  Wien.  med.  Wochenschr.,  190.3. 

'Zeitsehr.  f.  physiol.  Chem.,  24;  Pouchet,  "Contributions  a  la  connaissance  des 
mati^res  extractives  de  I'urine."  These  Paris,  1880.  Cited  from  Huppert-Neubauer, 
333  and  335. 

'  Deutsch.  med.  Wochensclir.,  1897. 


PURINE  BASES.  579 

Schmidt!  from  the  theobromine,  caffeine,  and  theophylhne  which  occur 
in  the  food.  "With  the  purine  bases  \xe  must  also  differentiate  between 
those  of  endogenous  and  those  of  exogenous  origin.-  As  the  four  true 
nuclein  bases  and  carnine  have  been  treated  in  Chapters  V  and  XI,  it  only 
remains  to  describe  the  special  urinarj'  purine  bodies. 

HX— CO 

I       I 
Heteroxanthine,    C6H6X402  =  7-monomethvlxanthine,    OC     C.X.CH,,  was  first 

detected  in  the  urine  by  Salomon.  It  is  identical  with  the  monomethvLxanthine 
which  passes  into  the  ui'ine  after  feeding  with  theobromine  or  caffeine.  Salomon^ 
and  Neuberg  *  found  heteroxanthine  in  the  urine  of  a  dog  fed  entirely  upon 
meat,  and  this  was  probably  formed  by  a  methylation  in  the  body. 

Heteroxanthine  crystallizes  in  .shining  needles  and  dissolves  with  difficulty 
in  cold  water  f]592  [^arts  at  1S°  C).  It  is  readily  soluble  in  amnionic,  and  alkaUes. 
The  crystalline  sodium  salt  Ls  insoluble  in  .strong  caustic  alkali  (.33  per  cent)  and 
dissolves  with  difficulty  in  water.  The  chloride  crystallizes  beautifully,  is  rela- 
tively insoluble,  and  is  readily  decomposed  into  the  free  base  and  hydiochloric 
acid  by  water.  Heteroxanthine  is  precipitated  by  copper  sulphate  and  bisul- 
phite, mercuric  chloride,  basic  lead  acetate  and  ammonia,  and  by  silver  nitr.ate. 
The  silver  compound  dissolves  rather  easily  in  dilute,  warm  nitric  acid;  it  crystal- 
lizes in  small  rhombic  plates  or  prisms,  often  grown  together,  forming  charac- 
teristic crosses.  Heteroxanthine  does  not  give  the  xanthine  reaction,  but  does 
give  \Yeidet/s  reaction,  accordins;  to  Fischer  (see  Chapter  V). 

CH3.X— CO 

i-Methylxanthine,   CeH^X^O, ,        OC     C.XH        ,  was   first    isolated   from  the 

1     ^!     ^CF 
HX-C.X.^-   ^' 

urine  and  studied  by  Kpuger,  and  then  by  Krt'ger  and  Salomon.^  It  is  diffi- 
cultly soluble  in  cold  water,  but  readily  soluble  in  ammonia  and  caustic  soda, 
and  does  not  give  an  insoluble  sodium  compound.  It  is  readily  soluble  in 
dilute  acids,  and  it  crystallizes  from  its  acetic-acid  solution  in  thin,  generally 
hexagonal  ])lates.  The  chloride  is  decomposed  into  the  base  and  hydrochloric 
acid  by  \s-ater.  1-methylxanthine  gives  crystalline  double  salts  vdth  platinum 
and  gold.  It  is  not  precipitated  by  ba.sic  lead  acetate,  nor  when  pure  by  basic 
lead  acetate  and  ammonia.  With  ammonia  and  silver  nitrate  it  gives  a  gela- 
tinous precipitate.  The  siivr-nitrate  compound  crystallized  from  nitric  acid 
forms  rosettes  of  united  needles.  With  the  xanthine  test  with  nitric  acid  it  gives 
an  orange  coloration  on  the  addition  oi  caustic  soda.  It  gives  Weidei.'s  reac- 
tion (according  to  Fiscuek)  beautifully. 


^  .Aibancse,  Ber.  d.  d.  chem.  Gesellsch.,  32;  Arch.  f.  exp.  Path.  u.  Pharm.,  35; 
Bondzynski  and  Gottlieb,  ibid.,  36,  and  Ber.  d.  deutsch.  chem.  GeseUsch.,  28;  E. 
Fischer,  ibid.,  30,  2405;  Kriiger  and  Salomon.  Zeitschr.  f.  physiol.  Chem.,  26:  Kriiger 
and  Schmidt,  Ber.  d.  d.  chem.  Gesellsch..  32,  and  Arch.  f.  exp.  Path.  u.  Pharm..  4o. 

-  See  Burian  and  Schur.  foot-note  3,  page  570,  and  Kaufmann  and  Mohr.  Deutsch. 
Arch.  f.  klin.  Med.,  74. 

'  Salkowski's  Festschrift,  1904. 

*  Kriiger,  Arch.  f.  (Anat.  u.)  Physiol.,  1894;  Kriiger  and  Salomon,  Zeitschr.  f 
physiol.  Chem.,  24. 


580  URINE. 

CH3.X— CO 
I      I 
Paraxanthine,         C7H8NP2  =  1.7-dimethylxanthine,      OC    C.X.CH3,    urotheo- 

HX— C.N^^" 

bromine  (Tiiudtchum),  was  first  isolated  from  the  urine  by  Thudichum  and  Sat.o- 
MON.'  It  crystallizes  beautifully  in  six-sided  plates  or  in  needles.  The  sodium 
compound  crystallizes,  in  rectangular  plates  oi-  prisms  and,  like  the  hetero- 
xanthine-sodium  compound,  is  insoluble  in  33  per  cent  caustic-soda  solution. 
The  sodium  compound  separates  in  a  crystalline  state  on  neutralizing  its  solution 
in  water.  The  chloride  is  readily  soluble  and  is  not  decomposed  by  water.  The 
chloroi:)latinate  crystallizes  very  beautifully.  ?^Iercuric  chloride  precipitates  it  only 
when  added  in  excess  and  after  a  long  time.  The  siher-nitrate  compound 
separates  as  white  silky  crystals  from  hot  nitric  acid  on  cooling.  It  gives  Weidel's 
reaction,  but  not  the  xanthine  test,  with  nitric  acid  and  alkali. 

Episarkine  is  the  name  given  by  Balke  to  a  purine  body  occurring  in  human 
urine.  The  same  body  has  been  observed  by  Salomon  ^  in  pigs'  and  dogs'  urine, 
as  well  as  in  urine  in  leucaemia.  Balke  gives  C^HoXgO  as  the  probable  formula 
for  episarkine.  It  is  nearly  insoluble  in  cold  water,  dissolves  with  difficulty  in 
hot  water,  but  may  be  obtained  therefrom  as  long  fine  needles.  Episarldne  does 
not  o-ive  the  xanthine  reaction  with  nitric  acid  nor  Weidel's  reaction.  With 
hydrochloric  acid  and  potassium  clilorate  it  gives  a  white  residue  which  turns 
violet  with  ammonia.  It  does  not  form  any  insoluble  sodium  compound.  The 
silver  compound  is  difficultly  soluble  in  nitric  acid.  Episarkine  is  possibly 
identical  with  epiiruanine. 

IIX—CO 

I    I 

Epiguanine,       CuH^XsO  =  7-methylguanine,  H2N.C    C.X.CH3,    was   first    pre- 

II       II         NflH 

pared  from  the  urine  by  Kruger.^  It  is  crystalline  and  difficultly  soluble  in 
hot  water  or  ammonia.  "  It  crystallizes  from  a  hot  33  per  cent  caustic-soda  solu- 
tion on  cooling  in  broad  shming  crystals  and  dissolves  readily  in  hydrochloric  or 
sulphuric  acid.  It  gives  a  characteristic  chloroplatinate  crystallizing  in  six-sided 
prisms.  It  is  precipitated  neither  by  basic  lead  acetate  nor  by  basic  lead  ace- 
tate and  ammonia.  Silver  nitrate  and  anmionia  give  a  gelatinous  precipitate. 
It  responds  to  the  xanthine  test  with  nitric  acid  and  alkali.  According  to 
Fischer  it  acts  like  episarkine  ^^ith  Weidel's  test. 

In  preparing  alloxuric  bases  from  the  urine,  the  fluid  is  supersaturated  with 
ammoiiia  and  precipitated  by  a  silver-nitrate  solution.  The  precipitate  is  then 
decomposed  with  sulphuretted  hydrogen.  The  boiling-hot  filtrate  is  evaporated 
to  ch-yncss  and  the  dried  residue  treated  with  3  per  cent  sulphuric  acid.  The 
purine  bases  are  dissolved,  while  the  uric  acid  remains  undissolved.  This  filtrate 
is  saturated  with  ammonia  and  precipitated  by  silver-nitrate  solution.  If  in- 
stead of  precipitating  with  silver  solution  we  desire  to  precipitate,  according  to 
Krijger  and  Wulff,  with  copi)er  suboxide,  the  urine  may  l)e  heated  to  boiling 
and  immediately  are  added,  successively,  100  c.c.  of  a  50  per  cent  sodium-bisul- 
phate  solution  and  100  c.c.  of  a  12  per  cent  copper-sulphate  solution  for  every 
liter  of  urine.  The  thoroughly  washed  precipitate  is  decomposed  with  hydro- 
chloric acid   and  sulphuretted  "hydrogen.     The   uric  acid  remains  in  great  part 

'Thudichum,  "Grundziige  d.  anal.  med.  klin.  Chemie"  (Berlin,  1886);  Salomon, 
Arch.  f.  (.\nat.  u.)  Physiol.,  1882,  and  Ber.  d.  deutsch.  chem.  Gesellsch.,  16  and  18. 

2  Balke,  "Zur  Kenntniss  der  Xanthinkorper"  (Inaug.-Diss.,  Leipzig,  1893);  Salo- 
mon, Zeitschr.  f.  physiol.  Chem.,  18. 

3  Arch.  f.  (Anat.  u.)  Physiol.,  1894;  Kriiger  and  Salomon,  Zeitschr.  f.  physiol. 
Chem.,  24  and  26. 


ESTIMATION  OF  ALLOXURIC   BASES.  581 

on  the  filter.     Further  details  in  regard  to  the  treatment  of  the  solution  of  the 
hydrochloric-acid  compounds  may  be  found  in  Kuuger  and  Salomon.^ 

Quarditatioe  Estimation  of  Allozuric  Bases  according  to  Salkowski.^ 
400  to  600  c.c.  of  the  urine  free  from  protein  is  first  precipitated  by  mag- 
nesia mixture  and  then  by  a  3  per  cent  silver-nitrate  solution  as  de- 
scribed on  page  577.  The  thoroughly  washed  silver  preicpitate  is  decom- 
posed by  sulphuretted  hydrogen  after  being  suspended  in  600-800  c.c. 
of  water  with  the  addition  of  a  few  drops  of  hydrochloric  acid.  It  is  heated 
to  boiling  and  filtered  hot,  and  finally  evaporated  to  dryness  on  the  water- 
bath.  The  residue  is  extracted  A\ith  20-30  c.c.  of  hot  3  per  cent  sulphuric 
acid  and  allowed  to  stand  twenty-four  hours;  the  uric  acid  is  filtered  off, 
washed,  the  filtrate  made  ammoniacal,  and  the  xantiiine  bodies  precipi- 
tated again  by  silver  nitrate,  the  precipitate  collected  on  a  small  chlorine- 
free  filter,  washed  thoroughly,  dried,  carefully  inciiierated,  the  ash  dis- 
solved in  nitric  acid,  and  titrated  with  ammonium  saij)hocyanide  accord' 
ing  to  Volhajrd's  method.  The  ammonium-sulphocyanide  solution  should 
contain  1.2-1.4  grams  per  liter,  and  its  strength  should  be  determined  by  a 
silver-nitrate  solution:  1  part  silver  corresponds  to  0.277  gram  nitrogen  of 
alloxuric  bases  or  to  0.7381  gram  alloxuric  b-awes.  By  this  method  the 
uric-acid  and  alloxuric  bases  can  be  simultaneously  determined  in  the  same 
portion  of  urine .^ 

Malfatti  ^  determines  the  nitrogen  of  the  allo>airic  bases  in  the  hydrochloric- 
acid  filtrate  from  the  separated  uric  acid.  This  nitrate  is  evaporated  with  mag- 
nesia until  all  the  ammonia  has  been  expelled  and  the  residue  used  for  the 
Kjeldahl  determination. 

The  nitrogen  of  the  alloxuric  bases  is  also  determined  as  the  difference  between 
the  uric-acid  nitrogen  and  the  total  nitrogen  of  the  alloxuric  bodies  of  the  silver 
precipitate  (Camerer,  Arnstein  ^).  Salkovvski  has  raised  the  objection  to 
this  procedure  that  it  is  not  possible  to  remove  all  the  ammonia  from  the  silver 
precipitate  by  washing.  According  to  Aknstein  *  this  can  readily  be  done  by 
boiling  the  precipitate  in  water  with  seme  magnesia,  and  under  these  circum- 
stances this  method  is  quite  serviceable.  The  nitrogen  is  estimated  by  Kjel- 
dahl's  method.  The  uric-acid  nitrogen  multiplied  by  3  gives  the  quantity  of 
uric  acid.  As  the  mixture  of  alloxuric  bases  in  the  urine  is  but  little  known,  the 
quantity  of  nitrogen  of  the  alloxuric  bases  is  always  calculated  as  a  certain 
alloxuric  base,  for  example  xanthine  (Camerer),  and  the  quantity  so  found 
used  as  a  measure  for  the  alloxuric  bases. 

According  to  a  new  method  of  Kruger  and  Schmid  '  the  uric  acid  and 
the  purine  bases  are  precipitated  as  a  cuprous  compound  by  copj^er-sulphate 
solution  and  sodium  bisulphite.  The  precipitate  is  decomposed  in  sufficient  water 
by  sodium  sulphide,  and  the  uric  acid  precipitated  from  the  concentrated  filtrate 
with  hydrochloric  acid,  and  the  purine  bases  again  precipitated  from  this  filtrate  as 

'  Zeitschr.  f.  physiol.  Chem.,  26,  and  also  Hoppe-Seyler-Thierf elder's  Handbuch, 
7,  Aufl.,  154. 

^  Pfliiger's  Arch.,  ('9. 

^  In  regard  to  the  details  we  refer  the  reader  to  the  original  paper. 

*  Centralbl.  f.  innere  Med.,  1897. 

5  Camerer,  Zeitschr.  f.  Biologie,  26  and  28;  Arnstein,  Zeitschr.  f.  physiol.  Chem.,  23. 

"  Salkowski,  1.  c;   Arnstein,  CentralU.  f.  d.  nied.  Wissensch.,  1S9S. 

'Zeitschr.  f.  physiol.  Chem.,  45'  and  Hoppe-Seyler-Thierfelder's  Handbuch, 
7.  Aufl.,  435. 


582  URINE. 

cuprous  or  silver  compounds.  Finally,  the  nitrogen  in  the  uric-acid  part  and  the 
part  containing  the  mixture  of  purine  bases  is  estimated. 

We  cannot  discuss  the  other  methods,  such  as  those  of  Deniges  and  Niemi- 
Lowicz,  and  the  method  suggested  by  Hall  '  for  clinical  purposes. 

Oxaluric  Acid,  CgH^Np,  =  (CON2H3).CO.COOH.  This  acid,  whose  relation 
to  uric  acid  and  urea  has  been  spoken  of  above,  does  not  always  occur  in  the 
urine,  and  then  only  in  traces  as  the  ammonium  salt.  This  salt  is  not  directly 
precipitated  by  CaClj  and  NHg,  but  on  boiling  it  is  decomposed  into  urea  and 
oxalate.  In  preparing  oxaluric  acid  from  urine  the  latter  is  filtered  through 
animal  charcoal.  The  oxalurate  retained  by  the  charcoal  may  be  obtained  by  boil- 
ing with  alcohol. 

COOH 
Oxalic  Acid,  C2H2O4,  or   -^^^t  occurs  under  physiological  conditions 

COOH 

in  very  small  amounts  in  the  urine,  about  0.02  gram  in  twenty-four  hours 
(FuRBRiNGER  2).  According  to  the  generally  accepted  view  it  exists  in 
tlie  urine  as  calcium  oxalate,  which  is  kept  in  solution  by  the  acid  phos- 
phates present.  Calcium  oxalate  is  a  frequent  constituent  of  urinary  sedi- 
ments and  occurs  also  in  certain  urinary  calculi. 

The  origin  of  the  oxalic  acid  in  the  urine  is  not  well  known.  Oxalic 
acid  when  administered  is  eliminated  unchanged,  at  least  in  part,  by  the 
urine  ;^  and  as  many  vegetables  and  fruits,  such  as  cabbage,  spinach, 
asparagus,  sorrel,  apples,  grapes,  etc.,  contain  oxalic  acid,  it  is  possible  that 
a  part  of  the  oxalic  acid  of  the  urine  originates  directly  from  the  food. 
That  oxalic  acid  may  be  formed  in  the  animal  body  as  a  metabolic  product 
from  proteins  or  fats  follows  from  the  observations  of  Mills  and  Luthje,* 
who  found  in  dogs  on  an  exclusively  meat  and  fat  diet,  as  also  in  starvation, 
that  oxalic  acid  was  eliminated  by  the  urine.  The  oxalic  acid  which  is 
eliminated  in  increased  quantity  with  a  diminished  oxygen  supply  and  an 
increased  protein  catabolism,  as  found  by  Reale  and  Boeri,  and  also  by 
Terray,  is  supposed  to  be  derived  partly  from  the  greater  destruction  of 
proteins.  Pure  protein  does  not,  according  to  Salkowski,^  increase  the 
quantity  of  oxalic  acid  eliminated;  on  the  contrary,  after  meat  feeding  the 
amount  of  this  acid  is  increased,  due  in  part  to  the  meat  containing 
oxalic  acid  (Salkowski).  Gelatine  and  gelatine-yielding  tissues  seem  to 
increase  the  excretion  of  oxalic  acid,  which  stands  in  accord  with  the 
observations  of  Kutscher  and  Schenk  6  that  on  the  oxidation  of  gelatine 
oxamic  acid  is  produced  from  the  glycocoll  and  this  then  decomposes 
readily  into  ammonia  and  oxalic  acid.     After  feeding  nucleins  no  constant 

'  Niemilowicz,  Zeitschr.  f.  physiol.  Chem.,  35;  Gittelmacher-Wilenko,  ibid.,  86; 
Hall,  Wien.  kl.n.  Wochenschr.,  16. 

^  Deutsch.  Arch.  f.  klin.  Med.,  IS.     See  also  Dunlop,  Journ.  Path,  and  Bacterid.,  3. 

^  In  regard  to  the  behavior  of  oxalic  acid  in  the  animal  body,  see  pages  629  and  6C0. 

^  Mills,  V;rchow's  Arch.,  99;    Liithje,  Zeitschr.  f.  klin.  Med.,  35. 

^  Reale  and  Boeri,  Wien.  med.  Wochenschr.,  1895;  Terray,  Pfliiger's  Arch.,  65; 
Salkowski,  Berl.  klin.  Wochenschr.,  1900. 

'Zeitschr.  f.  physiol.  Chem.,  43. 


OXALIC  ACID  AND   ALLAXTOIN.  583 

increase  in  the  elimination  of  oxalic  acid  lias  been  observed. ^  The  pro- 
duction of  oxalic  acid  due  to  an  incomplete  combustion  of  the  carbo- 
hydrates has  also  been  suggested.  The  work  of  Hildebraxdt  and  P. 
JMayer  seems  to  indicate  this  under  abnormal  conditions.  In  alimentary 
glycosuria  or  diabetes  Luzzato  2  could  not  observe  any  rise  in  the  elimi- 
nation of  oxalic  acid.  "We  have  no  grounds  for  the  assumption  that  oxalic 
acid  is  produced  under  physiological  conditions  by  an  incomplete  com- 
bustion of  carbohydrates.  We  cannot  exclude  the  possibility  of  the  for- 
mation of  oxalic  acid  from  the  oxidation  of  uric  acid  in  the  animal  body, 
yet  we  have  no  positive  proof  of  such  a  formation.-^ 

Oxalic  acid  is  best  detected  and  quantitatively  determined  according 
to  the  method  suggested  by  Salkowski:  Shaking  out  the  oxahc  acid  from 
the  acidified  urine  by  means  of  ether  and  then  proceeding  as  foUo-vrs  accord- 
ing to  AuTEXRiETH  and  Barth  :  -* 

The  twenty-four-hour  urine  is  precipitated  l:)y  CaCU  and  ammonia 
in  excess.  After  18-20  hours  the  precipitate  is  collected  (the  filtrate  must 
be  clear)  and  dissolved  in  a  Uttle  hydrochloric  acid  and  shaken  out  4-5 
times  vith  1.50-200  c.c.  ether  (containing  3  per  cent  absolute  alcohol). 
The  united  ethereal  extracts  are  filtered  through  a  dr\-  filter  and  distilled 
after  the  addition  of  about  5  c.c.  of  water.  The  liquid,  if  necessary-,  is 
decolorized  with  animal  charcoal  and  precipitated  with  CaClo  and  am- 
monia, mado  acid  after  a  certain  time  with  acetic  acid,  and  finally  the 
oxalate  is  collected,  washed,  burned  to  CaO,  and  weighed. 

.XH.CH.HN  V 
Allantoin  (Glyoxyldiureide),  C4H6N4O3 ,  0C<^         |  /CO,  oc- 

^XH.CO  HoX/ 
curs  in  the  urine  of  chiklren  within  the  first  eight  days  after  birth,  and  in  very 
small  amounts  also  in  the  urine  of  adults  (Gusserow,  Ziegler  and  Her- 
mann). It  is  found  in  rather  abundant  quantities  in  the  urine  of  pregnant 
women  (Gusserow).  Allantoin  has  also  been  found  in  the  urine  of  suck- 
ing calves  CW^ohler),  in  urine  of  oxen  (Salkowski),  and  sometimes  in  the 
urine  of  other  animals  (^Ieissxer).  It  is  also  found  in  the  amniotic  fluid 
and,  as  first  shown  by  Vauquelix  and  Lassaigxe,^  in  the  allantoic  fluid 
of  the  cow  (hence  the  name).  Allantoin  is  formed,  as  above  stated,  by  the 
oxidation  of  uric  acid  outside  of  the  animal  body,  hence  a  similar  formation 

'  See  Stradomsky,  Virchow's  -Vrch.,  1G3;  Mohr  and  Salomon,  Deutsch.  Arch.  f. 
klin.  Med.,  70;    Salkowski,  1.  c. 

^  Hildebrandt,  Zeitschr.  f.  physiol.  Chem.,  35;  P.  Mayer,  Zeitschr.  f.  klin.  Med.,  i'- 
Luzzato,  Salkowski's  Festschrift,  1904. 

^  See  "Wiener,  Lrgebnisse  der  Physiol.,  1,  Abt.  1. 

^  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  29;    Autenrieth  and  Barth,  ibid.,  35. 

5  Ziegler  and  Hermann,  see  Gusserow,  Arch.  f.  Gynjikol,  3 — both  cited  from  Huppert- 
Neubauer,  Harn-Analj^se,  10.  Aufl.,  377;  Wohler,  Annal.  d.  Chem.  u.  Pharm.,  70; 
Salkowski,  Zeitschr.  f.  physiol.  Chem.,  42;  Meissner,  Zeitschr.  f.  r.-.t.  Med.  (3),  31;  Las- 
saigne,  Annal.  de  Chim.  et  Phys.,  17. 


584  URINE. 

of  allantoin  is  assumed  in  the  animal  organism  (see  page  573).  According 
to  PoDUSCHKA  and  ]\Iinkowski,i  allantoin  introduced  into  dogs  appears 
almost  entirely  in  the  urine,  while  in  man  only  a  small  portion  of  the  in- 
gested substance  is  eliminated  by  the  kidneys.  In  carnivora  the  excretion 
of  allantoin  is  considerably  increased,  according  to  ^^Iinkowski,  Cohn, 
Salkowski,  and  ^Iendel  and  Brown,^  after  feeding  thymus  or  pancreas. 
According  to  Mendel  and  White,  on  the  intravenous  injection  of  urates 
an  abundant  elimination  of  allantoin  takes  place  in  dogs  and  cats.  A 
strong  allantoin  excretion  is  also  found  in  dogs  after  poisoning  with  hy- 
drazine (BoRissow),  hydroxy lamine,  semicarbazide,  and  aminoguanidine 
(PoHL^).  He  also  obtained  allantoin  in  the  autolysis  of  the  intestinal 
mucosa,  hver,  thymus,  spleen,  and  pancreas.  As  no  allantoin  exists  in 
the  organs  of  normal  starving  dogs,  and  as  Pohl  has  found  it  in  the  liver 
and,  as  traces,  also  in  the  other  organs  after  poisoning  with  hydrazine,  he 
claims  that  the  allantoin  is  formed  in  the  nuclein  destruction  produced 
by  the  death  of  the  cell-nuclei. 

Allantoin  is  a  colorless  substance  often  cr}^stallizing  in  prisms,  difficultly 
soluble  in  cold  water,  easily  soluble  in  boiling  water,  and  also  in  warm 
alcohol,  but  not  soluble  in  cold  alcohol  or  ether.  A  wateiy  allantoin  solu- 
tion gives  no  precipitate  with  silver  nitrate  alone,  but  by  the  careful  addi- 
tion of  ammonia  a  white  flocculent  precipitate  is  formed,  C4H^gN403, 
which  is  soluble  in  an  excess  of  ammonia  and  which  consists  after  a  certain 
time  of  ver}^  small,  transparent  microscopic  globules.  The  dry  precipitate 
contains  40.75  per  cent  silver.  A  water}-  allantoin  solution  is  precipitated 
by  mercuric  nitrate.  On  continued  boiling  allantoin  reduces  Fehling's 
solution.  It  gives  Schiff's  furfurol  reaction  less  rapidl}-  and  less  intensely 
than  urea.     Allantoin  does  not  give  the  murexide  test. 

Allantoin  is  most  easily  prepared  by  the  oxidation  of  uric  acid  with 
lead  peroxide.  In  preparing  allantoin  from  urine,  proceed  according  to 
Loewy's^  method,  which  consists  of  the  following:  The  faintly  acidified 
urine  is  precipitated  with  a  mercurous-nitrate  solution,  the  filtrate  treated 
with  H2S,  and  the  new  filtrate  precipitated  by  magnesium  oxide  and  silver 
nitrate  after  the  removal  of  the  H2S.  The  precipitate  is  filtered  off  and 
washed  with  warm  water  and  decomposed  with  H2S,  and  the  filtrate  evap- 
orated to  dryness.  The  residue  is  extracted  with  hot  water  and  then  the 
solution  precipitated  with  mercuric  nitrate.  The  precipitate  is  collected 
and   decomposed  by   H2S.     From   the   evaporated   filtrate   the   allantoin 

^  Arch.  f.  exp.  Path.  u.  Pharm.,  44;   Minkowski,  ibid.,  41. 

2  Minkowski,  1.  c,  and  Centralbl.  f.  innere  Med.,  1898;  Cohn,  Zeitschr.  f.  physiol. 
Chem.,  25;  Salkowski,  Centralbl.  f.  d.  med.  Wissenseh.,  1898;  Mendel  and  Brown, 
Amer.  Journ.  of  Physiol.,  3. 

^  Mendel  and  White,  Amer.  Journ.  of  Pliysiol.,  12;  Borissow,  Zeitschr.  f.  physiol. 
Chem.,  19;   Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.,  46. 

*  Arch.  f.  exp.  Path.  u.  P.  arm.,  44. 


HIPPURIC  ACID.  585 

ctystallizes  out.  This  method  can  be  used  for  the  quantitative  determina- 
tion of  allantoin. 

or*  n  TT 

Hippuric  Acid  (Benzoyl-amino  acetic  acid)  ,  C9H9NO0, 

^^  y.    9    9      ...  HN.CH2.COOH. 

This  acid  decom;.oses  into  benzoic  acid  and  glycocoll  on  boihng  with 
mineral  acids  or  alkaUes,  and  also  by  the  putrefaction  of  the  urine.  The 
reverse  of  this  occurs  if  these  two  components  are  heated  in  a  sealed  tube, 
according  to  the  following  equation:  C6H5COOH4-NH2.CH2.COOH  = 
C6H5.CO.NH.CH2.COOH  +  H2O.  This  acid  may  be  synthetically  pre- 
pared from  l)enzamide  and  monochloracetic  acid,  C6H5.CO.NH2  +  CH2CI. 
COOH  =  C6H5.CO.NH.CH2.COOH  +  HCl,  and  in  various  other  ways,  but 
most  simply  from  glycocoll  and  benzoyl  chloride  in  the  presence  of  alkali. 

Hippuric  acid  occurs  in  large  amounts  in  the  urine  of  herbivora,  but 
only  in  small  quantities  in  that  of  carnivora.  The  quantity  of  hippuric 
acid  eliminated  in  human  urine  on  a  mixed  diet  is  usually  less  than  1  gram 
per  day;  as  an  average  it  is  0.7  gram.  After  eating  freely  of  vegetables 
and  fruit,  especially  such  fruit  as  plums,  the  quantity  may  be  more  than 
2  grams.  Hippuric  acid  is  also  found  in  the  perspiration,  the  blood,  the 
suprarenal  capsule  of  oxen,  and  in  ichthyosis  scales.  Nothing  is  positively 
known  in  regard  to  the  quantity  of  hippuric  acid  in  the  urine  in  disease. 

The  Formation  of  Hippuric  Acid  in  the  Organism.  Benzoic  acid  and 
also  theisubstituted  benzoic  acids  are  converted  into  hippuric  acid  and  sub- 
stituted hippuric  acids  within  the  body.  Moreover,  those  bodies  are  trans- 
formed into  hippuric  acid  which  by  oxidation  (toluene,  cinnamic  acid, 
hydrocinnamic  acid)  or  by  reduction  (quinic  acid)  are  converted  into  ben- 
zoic acid.  The  question  of  the  origin  of  hippuric  acid  is  therefore  connected 
with  the  question  of  the  origin  of  benzoic  acid ;  the  formation  of  the  second 
component,  glycocoll,  frcta  the  protein  substances  in  the  body  is  unques- 
tionable. 

Hippuric  acid  is  found  in  the  urine  of  starving  dogs  (Salkoavski).  also 
in  dog's  urine  after  a  diet  consisting  entirely  of  meat  (^Ieissner  and 
Shepard,  Salkowski,  and  others  i).  It  is  evident  that  the  benzoic  acid 
originates  in  these  cases  from  the  proteins,  and  it  is  generally  admitted  that 
it  is  produced  by  the  putrefaction  of  proteins  in  the  intestine.  Amono-  the 
products  of  the  putrefaction  of  protein  outside  of  the  body  Salkowski  has 
found  phenylpropionic  acid,  C6H5.CH2.CH2.COOH,  which  is  oxidized  in 
the  organism  to  benzoic  acid  and  eliminated  as  hippuric  acid  after  combin- 
ing with  glycocoll.  Phenylpropionic  acid  seems  to  be  formed  from  the 
aminophenylpropionic  acid,  which  is  derived  from  several  protein  substances. 
The  supposition  that  the  phenylpropionic  acid  is  jiroduced  from  tvrosine  bv 


'  Salkowski,  Ber.  d.  dcutsch.  chem.  Gesellsch.,  11;    Meissncr  and  Shepard,  Unter- 
such.  iiber  das  Entstehen  der  Hippursaure  im  tliierischen  Organisnius.    Hanover  1866 


586  URINE. 

putrefaction  in  the  intestine  has  not  been  substantiated  by  the  researches 
of  Baumann,  Schotten,  and  Baas.i  The  importance  of  putrefaction  in 
the  intestine  in  producing  hippuric  acid  is  evident  from  the  fact  that  after 
thoroughly  disinfecting  the  intestine  of  dogs  with  calomel  the  hippuric  acid 
disappears  from  the  urine  (Baumann  2). 

The  large  quantity  of  hippuric  acid  present  in  the  urine  of  herbivora  is 
partly  explained  by  the  specially  active  processes  of  putrefaction  going  on 
in  the  intestines  of  these  animals,  but  it  is  especially  due  to  the  large  quantity 
of  substances  in  the  plant-food  from  which  benzoic  acid  can  be  formed. 
There  is  hardly  any  doubt  that  the  hippuric  acid  in  human  urine  after  a 
mixed  diet,  and  especially  after  a  diet  of  vegetables  and  fruits,  originates 
in  part  from  the  aromatic  substances,  e.g.,  quinic  acid. 

The  view  proposed  by  Weiss  and  others  that  a  parallelism  exists  between 
the  excretion  of  hippuric  acid  and  uric  acid  in  that  an  increase  in  the  first  is 
followed  by  a  diminution  in  the  second,  and  that,  for  example,  quinic  acid  produces 
a  diminution  in  the  excretion  of  uric  acid  corresponding  to  the  increased  forma- 
tion of  hippuric  acid  (Weiss,  Lewin),  cannot  be  considered  as  sufficiently  proved  * 
(Hupfer). 

The  kidneys  may  be  considered  in  dogs  as  special  organs  for  the  syn- 
thesis of  hippuric  acid  (Schmiedeberg  and  Bunge^).  In  other  animals 
as  in  rabbits,  the  formation  of  hippuric  acid  seems  to  take  place  in  other 
organs,  such  as  the  liver  and  muscles.  The  synthesis  of  hippuric  acid  is 
therefore  not  exclusively  limited  to  any  special  organ,  though  perhaps  in 
some  species  of  animals  it  may  be  more  abundant  in  one  organ  than  in 
another. 

As  the  thorough  investigations  of  Wiechowski  teach,  the  synthesis  of 
hippuric  acid  does  not  stand  in  any  direct  relationship  to  the  extent  of 
protein  metabolism;  it  varies,  on  the  contrary,  with  the  duration  of  circu- 
lation of  benzoic  acid  and  the  quantity  of  glycocoll  present  in  the  body. 
The  amount  of  the  latter  in  intermediary  metabolism  is  so  great  that  in 
rabbits,  on  the  administration  of  benzoic  acid,  more  than  one  half  of  the 
total  urine  nitrogen  may  exist  as  glycocoll.  ]Magnus-Levy  s  found  in 
rabbits  and  sheep  up  to  27.8  per  cent  of  the  total  nitrogen  as  hippuric-acid 

'  E.  and  H.  Salkowski,  Ror.  d.  deutsch.  chem.  Gesellsch.,  12;  Baumann,  Zeitschr. 
f.  physiol.  Chem.,  7;   Schotten,  ibid.,  8;   Baas,  ibid.,  11. 

'  Ibid.,  10,  131. 

3  Weiss,  Zeitschr.  f.  physiol.  Chem.,  25,  27,  3S;  Lewin,  Zeitschr.  f.  klin.  Med.,  42; 
Hupfer,  Zeitschr.  f.  physiol.  Chem.,  37.  See  also  Wiener,  "Die  Harnsiiure,"  Ergeb- 
nisse  der  Physiol.,  1,  Abt.  1. 

''  Arch.  f.  exp.  Path.  u.  Pharm.,  (»;  also  A.  Hoffmann,  ibid.,  7,  and  Kochs,  Pfliiger's 
Arch.,  20;   Bashford  and  Cramer,  Zeitschr.  f.  pliysiol.  Chem.,  35. 

^ Wiecliowski,  Hofmeister's  Beitrage,  7  (literature);  A.  Magnus- Levy,  Miinch. 
med.  Wochenschr.,  1905. 


PROPERTIES   AND  REACTIONS   OF  HIPPURIC   ACID.         587 

nitrogen,  and  both  investigators  have  found  so  much  hippuric-acid  nitrogen 
that  it  could  not  be  accounted  for  by  the  glycocoll  preformed  from  the 
proteins.  We  cannot  explain  how  this  aljundant  formation  and  eUmination 
of  glycocoll  as  hippuric  acid  is  brought  about. 

Properties  and  Reactions  of  Hippuric  Acid.  This  acid  cr}stallizes  in 
semi-transparent,  long,  four-sided,  milk-white,  rhombic  prisms  or  columns, 
or  in  needles  by  rapid  cr}-stallization.  They  dissolve  in  600  parts  cold 
water,  but  more  easily  in  hot  water.  They  are  easily  soluble  in  alcohol, 
but  with  difficulty  in  ether.  The  acid  dissolves  more  easily  (about  12  times) 
in  acetic  ether  than  in  ethyl  ether.  Petroleum-ether  does  not  dissolve 
hippuric  acid. 

On  heating  hippuric  acid  it  first  melts  at  187.5°  C.  to  an  oily  liciuid 
which  crystaUizes  on  cooling.  On  continuing  to  heat  it  decomposes,  pro- 
ducing a  red  mass  and  a  sublimate  of  l)enzoic  acid,  with  the  generation, 
first,  of  a  peculiar  pleasant  odor  of  hay  and  then  an  odor  of  hydrocyanic 
acid.  Hippuric  acid  is  easily  differentiated  from  benzoic  acid  by  this 
behavior,  also  by  its  crj^stalline  form  and  its  insolubility  in  petroleum 
ether.  Hippuric  acid  and  lienzoic  acid  both  give  Lucke's  reaction,  namely, 
they  generate  an  interse  odor  of  nitrol^enzene  when  evaporated  to  dr\'ness 
with  nitric  acid  and  when  the  residue  is  heated  with  sand  in  a  glass  tube. 
Hippuric  acid  forms  ciystalHzable  salts,  in  most  cases,  with  bases.  The 
comljinations  with  alkalies  and  alkaline  earths  are  soluble  in  water  and 
alcohol.  The  silver,  copper,  and  lead  salts  are  soluble  w^ith  difficulty  in 
water;  the  ferric  salt  is  insoluble. 

Hippuric  acid  is  best  prepared  from  the  fresh  urine  of  a  horse  or  cow. 
The  urine  is  boiled  a  few  minutes  with  an  excess  of  milk  of  lime.  The 
liquid  is  filtered  while  hot,  concentrated  and  then  cooled,  and  the  hippuric 
acid  precipitated  l^y  the  addition  of  an  excess  of  hydrochloric  acid.  The 
cr}'stals  are  pressed,  dissolved  in  milk  of  lime  by  boiling,  and  treated  as 
above;  the  hippuric  acid  is  precipitated  again  from  the  concentrated  fil- 
trate by  hydrochloric  acid.  The  cr}'stals  are  purified  by  recrs'stalUzation 
and  decolorized,  when  necessarj^,  by  animal  charcoal. 

The  quantitative  estimation  of  hippuric  acid  in  the  urine  may  be  per- 
formed by  the  following  method, (Bunge  and  Schmiedeberg  i):  The  urine 
is  first  made  faintly  alkaline  with  soda,  evaporated  nearly  to  dr}-ness.  and 
the  residue  thoroughly  extracted  with  strong  alcohol.  After  the  eva]3ora- 
tion  of  the  alcohol  the  residue  is  dissolved  in  water,  the  solution  acidified 
with  sulphuric  acid,  and  completely  extracted  by  agitating  (at  least  five 
times)  with  fresh  portions  of  acetic  ether.  The  acetic  ether  is  then  re- 
peatedly washed  with  water,  which  is  removed  by  means  of  a  separator}' 
funnel,  then  evaporated  at  a  medium  temperature  and  the  dr\'  residue 

*  Arch.  f.  exp.  Path.  u.  Pharm.,  6.  Tn  regard  to  other  methods,  such  as  Blumen- 
thal  as  well  as  Pfeiffer-,  Bloch  and  Riecke.  see  Maly's  Jahresber.,  30  and  32.  See  also 
Wiechowski,  1.  c. 


588  URINE. 

treated  repeatedly  with  petroleum-ether,  which  dissolves  the  benzoic  acid, 
oxyacids,  fats,  and  phenols,  while  the  hippuric  acid  remains  undissolved. 
This  residue  is  now  dissolved  in  a  little  warm  water  and  evaporated  at 
50-60°  C.  to  crystallization.  The  crystals  are  collected  on  a  small  weighed 
filter.  The  mother-liquor  is  repeatedly  shaken  with  acetic  ether.  This 
last  is  removed  and  evaporated ;  the  residue  is  added  to  the  above  crystals 
on  the  filter,  dried  and  weighed. 

Phenaceturic  Acid,  CioHuNOg  =CeH,.CH2.CO.NH.CH2.COOH.  This  acid,  which 
is  produced  in  the  animal  body  by  a  combination  of  glycocoll  with  the  phenyl- 
acetic  acid,  CHg.CHj.COOH,  formed  in  the  putrefaction  of  the  proteins,  has- 
been  prepared  from  horse's  urine  by  Sai^kowski,'  but  it  probably  also  occurs  in 
human  urine. 

Benzoic  Acid,  CyHr.Oj  or  CrHj.COOH,  is  found  in  rabbit's  urine  and  sometimes, 
though  in  small  amounts,  in  dog's  urine  (Weyl  and  v.  Anrep).  According  to 
Jaarsveld  and  Stokvis  and  to  Kronecker  it  is  also  found  in  human  urine  in 
diseases  of  the  kidneys.  The  occurrence  of  benzoic  acid  in  the  urine  seems  to 
be  due  to  a  fermentative  decomposition  of  hippuric  acid.  Such  a  decomposition 
may  very  easily  occur  in  an  alkaline  mine  or  in  one  containing  proteid  (Van  db 
Velde  and  Stokvis).  In  certain  animals — pigs  and  dogs — the  kidneys,  accord- 
ing to  ScHMiEDEBERG  and  MINKOWSKI,'  Contain  a  special  enzyme,  Schmiede- 
berg's  histozym,  which  s^Dlits  the  hippuric  acid  with  the  separation  of  benzoic 
acid. 

Ethereal  Sulphuric  Acids.  In  the  putrefaction  of  proteins  in  the  intes- 
tine, phenols,  whose  mother-substance  is  considered  to  be  tyrosine,  and  also 
indol  and  skatol  are  produced.  These  phenols  directly,  and  the  two  last- 
named  bodies  after  they  have  been  oxidized  respectively  into  indoxyl  and 
skatoxyl,  pass  into  the  urine  as  ethereal  sulphuric  acids  after  uniting  with 
sulphuric  acid.  The  most  important  of  these  ethereal  acids  are  phenol-  and 
cresol- sulphuric  acids — which  were  formerly  also  called  phenol-forming  sub- 
stances— indoxyl-  and  skatoxyl-suljyhuric  acids.  To  this  group  belong  also 
the  pyrocatechin-sidphuric  acid,  which  occurs  only  in  very  small  amounts  in 
human  urine,  and  hydroquinone-sulphuric  acid,  which  appears  in  the  urine 
after  poisoning  with  phenol,  and  under  physiological  conditions  perhaps 
other  ethereal  acids  occur  which  have  not  been  isolated.  The  ethereal 
sulphuric  acids  of  the  urine  were  discovered  and  specially  studied  by  Bau- 
MANN.^  The  quantity  of  these  acids  in  human  urine  is  small,  while 
horse's  urine  contains  larger  quantities.  According  to  the  determinations 
of  V.  D.  Velden  the  quantity  of  ethereal  sulphuric  acid  in  human  urine  in 
twenty-four  hours  varies  between  0.094  and  0.620  gram.  The  relationship 
of  the  sulphate-sulphuric  acid  A  to  the  conjugated  sulphuric  acid  B  in 


'  Zeitschr.  f.  physiol.  Chem.,  9. 

^Weyl  and  v.  Anrep,  Zeitschr.  f.  physiol.  Chem.,  4;  Jaarsveld  and  Stokvis,  Arch, 
f.  exp.  Path.  u.  Pharm.,  10;  Kronecker,  ibid  ,  16;  "Van  der  Velde  and  Stokvis,  ibid., 
17;    Schmiedeberg,  ibid.,  14,  379;    Minkowski,  ibid.,  17. 

3Pfliiger's  Arch.,  12  and  13. 


PHENOL   AND   P-CRESOL-SULPHURIC   ACID.  589 

health  is  on  an  average  as  10:1.  It  undergoes  such  great  variations,  as 
found  by  Bau^l^xx  and  Herter.^  and  after  them  by  many  other  investi- 
gators, that  it  is  hardly  possible  to  consider  the  average  figures  as  normal. 
After  taking  phenol  and  certain  other  aromatic  substances,  as  well  as  -when 
putrefaction  within  the  organism  is  general,  the  eUmination  of  ethereal 
sulphuric  acid  is  greatly  increased.  On  the  contrar\-,  it  is  diminished  when 
the  jDutref action  in  the  intestine  is  reduced  or  prevented.  For  this  reason  it 
may  be  greatly  diminished  by  carbohydrates  and  exclusive  milk  diet.^  The 
intestinal  putrefaction  and  the  elimination  of  ethereal  sulphuric  acid  have 
also  been  diminished  in  some  cases  by  certain  therapeutic  agents  which 
have  an  antiseptic  action;  still  the  statements  are  not  unanimous.^ 

Great  importance  has  been  given  to  the  relationship  between  the  total 
sulphuric  acid  and  the  conjugated  sulphuric  acid,  or  between  the  conjugated 
sulphuric  acid  and  the  sulphate-.sulphuric  acid,  in  the  study  of  the  intensity 
of  the  putrefaction  in  the  intestine  under  different  conditions.  Several 
investigators,  F.  Muller,  Salkowski.  and  v.  Noordex.^  consider  cor- 
rectly that  tliis  relationship  is  only  of  secondaiy  value,  and  that  it  is  more 
correct  to  consider  the  absolute  value.  It  must  be  remarked  that  the  abso- 
lute values  for  the  conjugated  sulphuric  acid  also  undergo  great  variation, 
so  that  it  is  at  present  impossible  to  give  the  upper  or  lower  limit  for  the 
normal  value. 

Phenol-  and  p - Cresol-sulphuric  Acids,  C6H5.O.SO2.OH  and 

C6H4<qtV'    "■       .     These  acids  are  found  as  alkaU  salts  in  human  urine, 

in  which  also  orthocresol  has  been  detected.  The  quantity  of  cresol-sul- 
phuric acid  is  considerably  greater  than  of  phenol-sulphuric  acid.  In  the 
quantitative  estimation  the  phenols  are  set  free  from  the  two  ethereal  acids 
and  determined  together  as  tribromphenol.  The  quantity  of  phenols  which 
are  separated  from  the  ethereal-sulphuric  acids  of  the  urine  amounts  to  17-51 
milhgrams  in  the  twenty -four  hours  (:\Iuxk).  The  methods  for  the  quanti- 
tative estimation  used  heretofore  give,  according  to  Ru.mpf.  as  well  as  Koss- 
LER  and  Pexxy.^  such  inaccurate  results  that  new  determinations  are  ver>' 
desiral)le.     .After  a  vegetable  diet  the  quantity  of  the.se  ethereal-sulphuric 


M-.  d.  Velden.  Viichow's  Arch.,  70;   Herter,  Zeitschr.  f.  physiol.  Chem.,  1. 

2  See  Hirscliler,  Zeitschr.  f.  physiol.  Chem.,  10;  Biernacki,  Deutsch.  Arch.  f.  khn. 
Med.,  -49;  Rovighi,  Zeitschr.  f.  physiol.  Chem.,  16;  "Winternitz,  ibid.,  and  Sclmiitz, 
ibid.,  17  and  19. 

^  See  Baumann  and  Morax,  Zeitschr.  f.  physiol.  Chem.,  10;  Steiff,  Zeitschr.  f. 
khn.  Med.,  16;  Ro\-igVi.  1.  c,  Stem,  Zeitschr.  f.  Hygiene,  12;  and  Bartoschewitsch, 
Zeitschr.  f.  physiol.  Chem.,  1";    Mosse,  ibid..  23. 

*Muller,  Zeitschr.  f.  khn.  Med.,  12;  v.  Xooiden,  ibid.,  17;  Salkowski,  Zeitschr. 
f  physiol.  Chem.,  12. 

^Munk,  Pfliiger's  Arch.,  12;  Rumpf,  Zeitschr.  f.  physiol.  Chem.,  16;  Ivossler  and 
Penny,  ibid.,  17. 


690  URINE. 

acids  is  greater  than  after  a  mixed  diet.  After  the  ingestion  of  carbolic  acid, 
which  is  in  great  part  converted  by  sj-nthesis  within  the  organism  into  phe- 
nol-sulphuric acid,  also  into  pyrocatechin-  and  hydroquinon-sulphuric  acid,^ 
or  when  the  amount  of  sulphuric  acid  is  not  sufficient  to  combine 
with  the  phenol,  it  forms  phenyl-glucuronic  acid  ^  the  quantit}^  of  phenols 
and  ethereal-sulphuric  acids  in  the  urine  is  considerably  increased  at  the 
expense  of  the  sulphate-sulphuric  acid. 

An  increased  elimination  of  phenol-sulphuric  acids  occurs  in  active 
putrefaction  in  the  intestine  with  stoppage  of  the  contents  of  the  intestine, 
as  in  ileus,  diffused  peritonitis  with  atony  of  the  intestine,  or  tuberculous 
enteritis,  but  not  in  simple  obstruction.  The  elimination  is  also  increased 
by  the  absorption  of  the  products  of  putrefaction  from  purulent  wounds  or 
abscesses.  An  increased  elimination  of  phenol  has  bee<n  oljserved  in  a  few 
other  cases  of  diseased  conditions  of  the  body.^ 

The  alkali  salts  of  phenol-  and  cresol-sulphuric  acids  crystallize  in  white 
plates,  similar  to  mother-of-pearl,  which  are  rather  freety  soluble  in  water. 
They  are  soluble  in  boiling  alcohol,  but  only  slightly  soluble  in  cold  alcohol. 
On  boiling  with  dilute  mineral  acids  they  are  decomposed  into  sulphuric 
acid  and  the  corresponding  phenol. 

Phenol-sulphuric  acids  have  been  synthetically  prepared  by  Baumann 
from  ]:>otassium  pyrosvilphate  and  potassium  phenolate  or  p-cresolate.  For 
the  method  of  their  preparation  from  urine,  which  is  rather  complicated, 
and  also  for  the  known  ])henol  reactions,  the  reader  is  referred  to  other 
text-l)ooks.  The  quantitative  estimation  of  these  ethereal-sulphuric  acids 
was  usualty  performed  by  weighing  the  phenol  which  was  separated  from 
the  urine  as  tribromphenol.  At  the  present  time  the  following  method  is 
employed : 

KossLER  and  Penny's  Method  with  Neuberg's^  Modification.  The 
liquid  containing  phenol  is  treated  with  N/10  caustic  soda  until  strongly 
alkaline,  warmed  on  the  water-l^ath  in  a  flask  with  a  glass  stopper,  and 
then  treated  with  an  excess  of  N/10  iodine  solution,  the  quantity  being 
exactly  measured.  Sodium  iodide  is  first  formed  and  then  sodium  hypo- 
iodite.  which  latter  forms  tri-iodophenol  with  the  phenol  according  to  the 
following  equation: 

CeHsOH  +  3NaI0  =  CeHoIg.OH  +  3NaOH. 

On  cooling  acidify  with  sulphuric  acid  and  determine  the  excess  of  iodine 
by  titration  with    N/10    sodium   thiosulphate    solution.     This   process  is 

'  See  Baumann,  Pfliiger's  Arch.,  12  and  13,  and  Baumann  and  Preusse,  Zeitsehr. 
f.  physiol.  Chem.,  3,  156. 

^  Sf^bmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  14. 

^  See  G.  Hoppe-Seyler,  Zeitsehr.  f.  physiol.  Chem.,  12,  (this  contains  also  all 
references  to  the  literature  on  this  subject  ;  Fedeli,  Moleschott's  Untensuch.,  15. 

*  Koss'.er  and  Penny,  Zeitsehr.  f.  physiol.  Chem.,  1";    Neuberg,  ibid.,  27. 


PYROCATECHIN  AND  HYDROQUINONE.  591 

also  available  for  the  estimation  of  paracresol.  Each  cubic  centimeter 
of  the  iodine  solution  used  is  equivalent  to  1.5670  milligrams  of  phenol  or 
1.8018  miUigrams  of  cresol.  As  the  determination  does  not  give  any  idea 
as  to  the  variable  proportions  of  the  two  phenols,  the  quantity  of  iodine 
used  must  be  calculated  as  one  or  the  other  of  the  two  phenols.  Before 
such  a  determination  is  carried  out,  the  concentrated  urine  is  first  distilled 
after  acidification  with  sulphuric  acid  and  the  distillate  purified  by  pre- 
cipitation with  lead  and  distilled  again  (Neuberg).  For  details,  see 
Neuberg,  1.  c,  and  Hoppe-Seyler-Thierfelder's  Handbuch,  7.  Aufl. 

The  methods  for  the  separate  determination  of  the  conjugated  sulphuric 
acid  and  the  sulphate-sulphuric  acid  will  be  spoken  of  later  in  connection 
with  the  determination  of  the  sulphuric  acid  of  the  urine. 

Pyrocatechin-sulphuric  Acid.  This  acid  was  first  found  in  horse's  urine  in 
rather  large  quantities  by  Baumann.  It  occurs  in  human  urine  only  in  the 
very  smallest  amounts,  and  perhaps  not  constantly,  but  it  is  present 
abundantly  in  the  urine  after  taking  phenol,  pyrocatechin,  or  protocatechuic 
acid. 

With  an  exclusively  meat  diet  this  acid  does  not  occur  in  the  urine,  and  it 
therefore  must  originate  from  vegetable  food.  It  probably  originates  from  the 
protocatechuic  acid,  which,  according  to  Preusse,  passes  in  part  into  the  urine 
as  pyrocatechin-sulphuric  acid.  This  acid  may  also  perhaps  be  formed  by  the 
oxidation  of  phenol  within  the  organism  (Baumann  and  Preusse  '). 

Pyrocatechin,  or  o-I^ioxybenzene,  CHJOH)^,  was  first  observed  in  the  urine 
of  a  child  (Ebstein  and  J.  Mult.er).  The  reducing  body  alcapton,  first  found 
by  Bodeker  -  in  human  urine  and  which  was  considered  for  a  long  time  as  iden- 
tical with  pyrocatechin,  is  in  most  cases  probably  homogentisic  acid  or  urolcucic 
acid  (see  below). 

Pyrocatechin  crystallizes  in  prisms  which  are  soluble  in  alcohol,  ether,  and 
water.  It  melts  at  102-104°  C,  and  sublimes  in  shining  plates.  The  watery 
solution  becomes  green,  brown,  and  ultimately  black  in  the  presence  of  alkali  and 
the  oxygen  of  the  air.  If  very  dilute  ferric  chloride  is  treated  with  tartaric  acid 
and  then  made  alkaline  with  ammonia,  and  this  added  to  a  watery  solution 
of  pjTocatechin,  we  obtain  a  violet  or  cherry-red  liquid  which  becomes  green 
on  adding  excess  of  acetic  acid.  Pyrocatechin  is  precipitated  by  lead  acetate. 
It  reduces  an  ammoniacal  silver  solution  at  the  ordinary  temperature,  and  re- 
duces alkaline  copper-oxide  solutions  with  heat,  but  does  not  reduce  bismuth 
oxide. 

A  urine  containing  pyrocatechin,  if  exposed  to  the  air,  especially  when  alkaline, 
quickly  becomes  dark  and  reduces  alkaline  copper  solutions  when  heated.  In 
detecting  pyrocatechin  in  the  urine  it  is  concentrated  when  necessary,  filtered, 
boiled  with  the  addition  of  sulphuric  acid  to  remove  the  phenols,  and  repeatedly 
shaken  after  cooling  with  ether.  The  ether  is  distilled  from  the  several  ethereal 
extracts,  the  residue  neutralized  with  barium  carbonate  and  shaken  again  with 
ether.  The  pyrocatechin  which  remains  after  evaporating  the  ether  may  be 
purified  by  recrystallization  from  benzene. 

Hydroquinone,  or  p-Dioxybenzene,  CH^COH),,  often  occurs  in  the  urine  after 
the  use  of  phenol  (Baumann  and  Preusse).  The  dark  color  which  certain  urines, 
so-called  "carbolic  urines,"  assume  in  the  air  is  due  to  decomposition  products'. 
Hydroqinnone  does  not  occur  as  a  normal  constituent  of  urine,  but  only  after 
the  administration  of  hydroquinone;  and  according  to  Lewin,^  it  may  be  found 

'  Baumann  and  Herter,  Zeitschr.  f.  physiol.  Cheni.,  1;  Preusse,  ibid.,  2;  Baumann 
ibid.,  3. 

2  Ebstein  and  Miiller,  Virchow's  Arch.,  62;  Bodeker,  Zeitschr.  f.  rat.  Med.  (3)^  7. 
^  Vircliow's  Arch.,  92. 


592  URINE. 

in  the  urine  of  rabbits  as  an  ethereal-sulphuric  acid,  being  a  decomposition 
product  of  arbutin.  .  ,   ,  ,     . 

Hydroquinone  forms  rhombic  crystals  which  are  readily  soluble  in  water, 
alcohol,  and  ether.  It  melts  at  169°  C.  Like  pyrocatechin,  it  easily  reduces 
metallic  oxides.  It  acts  like  pjTOcatechin  with  alkalies,  but  is  not  precipitated 
with  lead  acetate.  It  is  oxidized  into  quinone  by  ferric  cliloride  and  other  oxidiz- 
ing agents,  and  quinone  can  be  detected  by  its  peculiar  odor.  Hydroquinone- 
sulphuric  acid  is  detected  in  the  m'ine  by  the  same  methods  as  pyrocatecliin- 
sulphuric  acid. 

CH 

/\ 
Indoxyl-sulphuric  Acid,  C8H7NS04  =  HC      C— C.O.SOgCOH),  also  called 

I        II     II 
HC      C    CH 

X/\/ 
CH  NH 

URINE  INDICAN,  formerly  called  uroxaxthine  (Heller),  occurs  as  an  alkali- 
salt  in  the  urine.  This  acid  is  the  mother-substance  of  a  great  part  of 
the  indigo  of  the  urine.  The  quantity  of  indigo  Vv'hich  can  be  separated 
from  the  urine  is  considered  as  a  measure  of  the  quantity  of  indoxyl-sul- 
phuric acid  (and  indoxyl-glucuronic  acid)  contained  in  the  urine.  This 
amount,  according  to  Jaff^;,^  for  man  is  5-20  milligrams  per  twenty -four 
hours.  Horse's  urine  contains  about  twenty-five  times  as  much  indigo- 
forming  substance  as  human  urine. 

Indoxyl-sulphuric  arid  is  derived,  as  previously  mentioned  (page  401), 
from  indol,  which  is  first  oxidized  in  the  body  into  indoxyl  and  is  then 
conjugated  with  sulphuric  acid.  After  subcutaneous  injection  of  indol  the 
elimination  of  indican  is  considerabty  increased  (Jaffe,  Baumann  and 
Brieger,  and  others).  It  is  also  increased  by  the  introduction  of  orthoni- 
trophenylpropioUc  acid  in  the  animal  organism  (G.  Hoppe-Seyler^). 
Indol  is  formed  by  the  putrefaction  of  proteins.  The  putrefaction  of  se- 
cretions rich  in  protein  in  the  intestine  explains  also  the  occurrence  of  indican 
in  the  urine  during  starvation.  Gelatine,  on  the  contrary,  does  not  increase 
tlie  elimination  of  indican. 

An  abnormally  increased  elimination  of  indican  occurs  in  those  diseases 
where  the  small  intestines  are  obstructed,  causing  an  increased  putrefaction 
and  thus  producing  an  al^undance  of  indol.  Such  an  increased  elimination 
of  indican  occurs  on  tying  the  small  intestine  of  a  dog,  but  not  the  large 
intestine  (Jaffe),  an  observation  which  has  been  confirmed  recently  by 
Ellinger  and  Prutz.^  They  removed  an  intestinal  loop  in  dogs  and 
replaced  it  in  a  reversed  position,  the  distal  end  of  the  loop  being  attached 

1  Pfliiger's  Arch.,  3. 

^  Jaff6,  Centralbl.  f.  d.  med.  Wissensch.,  1872;  Baumann  and  Brieger,  Zeitschr.  f. 
physiol.  Chem.,  3;  G.  Hoppe-Seyler,  ibid.,  7  and  8.  See  also  Porcher  and  Ilervieux, 
Joum.  de  Physiol.,  7. 

'  Jaffe,  Virchow's  Arch.,  70;   Ellinger  and  Prutz,  Zeitschr.  f.  physiol.  Chem.,  38. 


INDOXYL-SULPHURIC  ACID.  593 

to  the  proximal  end  of  the  intestine,  and  in  this  manner,  by  the  inverted 
peristalsis  so  obtained,  they  effected  a  disturbance  in  the  movement  of  the 
intestinal  contents.  It  was  shown  that  this  obstruction  in  the  small  intes- 
tine caused  an  increased  elimination  of  indican,  wliile  an  obstruction  in  the 
large  intestine  showed  no  such  action. 

The  putrefaction  of  proteins  in  other  organs  and  tissues  besides  the  in- 
testine may  also  cause  an  increase  in  the  indican  of  the  urine.  Certain 
investigators.  Blumexthal.  Rosenfeld,  and  Lewix.  claim  to  have  shown 
that  an  increased  excretion  of  indican  can  be  l:)rought  about  also  \\  ithout 
putrefaction  by  an  increased  destruction  of  tissue  in  starvation  and  also 
after  i)hlorhizin  poisoning;  but  these  statements  are  vehemently  opposed 
by  other  investigators,  such  as  P.  Mayer,  Scholz.  and  Ellixger.  and  are 
improbable.  The  indol.  it  seems,  is  not  formed  from  the  tryi^tophane 
(indolaminopropionic  acid)  as  intermediary"  step  in  the  demolition  of  the 
proteins  in  the  animal  body,  but  rather  from  the  ])utrefaction  of  the  tri'pto- 
phane  in  the  intestine.  Gextzex'  ^  has  also  shown  that  tryptophane  intro- 
duced subcutaneously  or  per  os  into  the  body  does  not  lead  to  an  indican- 
uria,  but  only  when  it  is  exposed  to  bacterial  decomposition  in  the  large 
intestine.  The  statements  as  to  the  eUmination  of  indican  after  oxalic- 
acid  poisoning  are  somewhat  contradictoiy.  After  poisoninc;  with  oxalic 
acid  Harxack  and  v.  Leyex  found  an  increased  indican  elimination,  and 
MoRACZEWSKi  believes  he  has-  proved  a  certain  parallelism  between  the 
quantity  of  indican  and  the  quantity  of  oxalic  acid  in  diabetes.  Scholz  - 
obtained,  on  the  contrary,  no  increase  in  the  excretion  of  indican  after 
oxalic-acid  poisoning. 

An  increased  eUmination  of  indican  has  been  observ-ed  in  manv  dis- 
eases.^ and  in  these  cases  the  quantity  of  phenol  eliminated  is  also  gener- 
ally increased.     A  urine  rich  in  phenol  is  not  always  rich  in  indican. 

The  potassium  salt  of  indoxyl-sulphuric  acid,  wliich  was  prepared  pure 
by  Bau^l^xx  and  Brieger  from  the  urine  of  a  dog  fed  on  indol.  has  since 
been  prepared  synthetically  V)y  Baumaxx  and  Thesex.-*  by  fusing  phenyl- 
glycine-orthocarboxylic  acid  with  aDcaU  and  then  from  tliis  producing  the 

>BIumenthal.  -\rch.  f.  (Anat.  u.)  Physiol.,  1901,  Suppl.,  and  1902,  with  Rosenfeld, 
Charite  annaleu.  27.  and  Hofmeister's  Reitrage.  o:  Lewin,  Hofmeister's  Beitrage.  1; 
Mayer.  Arch.  f.  (.\nat.  u.)  Physiol.,  1902.  Zeitschr.  f.  klin.  Med.,  47.  and  Zeitsclir.  f. 
physiol.  Chem.,  29,  32;  Scholz,  ibid..  3S;  Ellinger,  ibid.,  39;  Gentzen.  "ITjer  die  Vors- 
tufen  des  Indols  bei  der  Eiweissfaulnis  im  Thierkorper,"  Inaug. -Dissert.  Koncisberg, 
1901. 

2  Hamack,  Zeitschr.  f.  physiol.  chemie,  29;  Scholz,  1.  c:  Moraczewski.  Centralbl. 
f.  innere  Med.,  1903. 

3  See  Jaffe.  Pfliiger's  Arch.,  3;  Senator,  Centralbl.  f.  d.  med.  Wissensch..  1S77; 
G.  Hoppe-Seyler.  Zeitschr.  f.  physiol.  Chem..  12  (contains  older  literature);  also 
Berl.  klin.  Wochenschr.,  1S92. 

*  Baumann  with  Brieger,  Zeitschr.  f.  physiol.  Chem.,  3;    with  Thesen.  ibid.    23. 


594  URINE. 

indoxylsulphate  by  means  of  potassium  pyrosulphate.  It  crystallizes  in 
colorless,  shining  plates  or  leaves  which  are  easily  soluble  in  water,  but  less 
readily  in  alcohol.  It  is  split  by  mineral  acids  into  sulphuric  acid  and 
indoxyl.  The  latter  without  access  of  air  passes  into  a  red  compound 
indoxyl-red,  ])ut  in  the  presence  of  oxidizing  reagents  is  converted  into 
indigo-blue:  2C8H7NO  +  20=Ci6HioN202  +  2H20.  The  detection  of  indi- 
can  is  based  on  this  last  fact. 

For  the  rather  complicated  preparation  of  indoxyl-sulphuric  acid  as  the 
potassium  salt  from  urine  the  reader  is  referred  to  other  text-books.  For 
the  detection  of  indican  in  urine  in  ordinary'  cases  the  following  method  of 
Jaffe-Obermayer,  wliich  also  serves  as  an  approximate  test  for  the  quan- 
tity of  indican,  is  sufficient. 

JaffiS-Obermayer's  Indican  Test.  Jaffe  uses  chloride  of  lime  as  the 
oxidizing  agent,  while  Obermayer  employs  ferric  chloride.  Other  oxidizing 
agents  have  been  suggested,  such  as  potassium  permanganate,  potassium 
bichromate,  alkali  chlorate,  and  hydrogen  peroxide  (the  latter  suggested 
by  Porcher  and  HervieuxI).  With  Obermayer's  reagent  the  test  is 
performed  as  follows: 

The  acid  urine  (if  alkaline  it  must  l;)e  acidified  with  acetic  acid)  (Ellin- 
ger)  is  precipitated  with  Ijasic  lead  acetate,  1  c.c.  for  every  10  c.c.  of 
the  urine.  20  c.c.  of  the  filtrate  are  treated  in  a  test-tube  with  an  equal 
volume  of  pure  concentrated  hydrochloric  acid  (specific  gra\dty  1.19) 
which  contains  2-4  grams  ferric  chloride  to  the  liter,  and  2-3  c.c.  chloro- 
form are  added  and  the  mixture  immediately  thoroughly  shaken.  The 
chloroform  is  thereby  colored  more  or  less  blue,  depending  upon  the 
amount  of  indican.  Besides  indigo  blue  we  may  also  have  indigo  red  pro- 
duced, whose  formation  has  been  explained  in  various  ways.  The  quantity 
of  indigo  red  l^ecomes  greater  the  more  slowly  the  oxidation  takes  place, 
and  especially  when  the  decomposition  takes  place  in  the  warmth  (see  the 
works  of  Rosin,  Bouma,  Wang,  Maillard,  and  Ellinger^). 

According  to  Ellinger  the  source  of  the  indigo-red  formation  may  be  the 
isatin  that  is  produced  by  the  superoxidation  of  the  indoxj^  by  the  action  of 
the  reagent,  and  this  isatin  forms  indigo  red  with  the  indoxyl  in  the  hydrochloric- 
acid  solution.  Maillard,  on  the  contrarj',  is  of  the  view  that  the  blue  substance 
which  is  taken  up  by  the  chloroform  from  the  urine  mixed  with  hydrochloric 
acid  is  not  indigotin  (indigo-blue),  but  another  substance,  called  by  him  hemi- 
indigotin,  which  in  alkaline  solution  polymerizes  immediately  into  indigotin, 
while  in  acid  reaction  it  is  converted  into  indirubin  (indigo  red). 

The  chloroform  solution  of  indigo  obtained  in  the  indican  test  may  be 
used  in  the  quantitative  colorimetric  determination  l)y  comparison  with  a 
solution  of  indigo  in  chloroform  of  known  strength  (ICrauss  and  Adrian)  . 

'  Jaffe,  Pfliiger's  Arch.,  3;  Obermayer,  Wien.  klin.  Wochenschr.,  1890;  Porcher 
and  Hervieux,  Zeitschr.  f.  physiol.  Chem.,  39. 

^  Rosin,  Virchow's  Arch.,  123;  Bouma,  Zeitschr.  f.  pliysiol.  Chem.,  27,  30,  82, 
39;  Wang,  ibid.,  25,  2",  28;  Ellinger,  ibid.,  3S  and  -11;  Maillard,  Bull.  soc.  chim.,  Paris 
(.3),  29,  and  Compt.  rend.,  36;  also  L'indoxyle  urinaire  et  les  couleurs  qui  en  derivent, 
Paris,  190.3,  and  Zeitschr.  f.  physiol.  Chem.,  41. 


SKATOXYL-SULPHURIC  ACID.  595 

Wang  and  others  convert  the  indigo  into  indigo-sulphonic  acid  by  con- 
centrated siilphuriq  acid  and  titrate  with  potassium  permanganate.  There 
is  still  doubt  as  to  the  surest  and  most  trustwortlw  method  for  the  deter- 
mination of  indican,  and  especially  as  to  the  question  how  the  indigo  resi- 
due is  to  be  washed  (see  Wang,  Bou>l\,  Ellinger,  and  Salkowski  ^),  and 
for  this  reason  we  shall  refer  only  to  the  works  cited  above. 

Because  of  the  difficulty  arising  from  the  production  of  indirul)in  in 
addition  to  indigotin,  Bouma  has  recommended  the  conversion  of  all  the 
indoxyl  into  indirubin  by  boiling  the  urine  with  hydrochloric  acid  contain- 
ing isatin.  The  indiruljin  can  l^e  taken  up  by  chloroform  and  deter- 
mined by  titration  with  potassium  permanganate  and  sulphuric  acid  after 
purification  of  the  chloroform  residue.  Oerum  ^  has  also  worked  out  a 
colorimetric  method  of  estimation  based  upon  Bouma's  method. 

Indol  seems  also  to  pass  into  the  urine  as  a  glucuronic  acid,  indoxyl- 
giucuronic  acid  (Schmiedeberg).  Such  an  acid  has  been  found  in  the  urine 
of  animals  after  the  administration  of  the  sodium-salt  of  o-nitro-phenyl- 
propiohc  acid  (G.  Hoppe-Seyler).  Porcher  and  Hervieux  ^  have  ob- 
tained indoxyl  sulphuric  acid  in  dogs  and  asses  under  similar  conditions. 

Free  indigo,  and  in  fact  indirubin  as  well  as  indigotin,  occur  in  rare  cases 
in  the  undecomposed  urine.  Grober  and  W.\ng  *  have  recently  observed  such 
cases. 

CH 

/\ 
HC      C— C.CH3 
Skatoxyl-sulphuric   Acid,    C9H9NS04=     |        ||     ||  ,  has  not 

HC      C    C.O.SOoOH 


CH  NH 
been  positively  prepared  as  a  constituent  of  normal  urine,  but  Otto  has 
once  prepared  its  alkali  salt  from  dialjetic  urine.  Perhaps  skatoxyl  occurs 
in  normal  urine  as  a  conjugated  glucuronate  (Mayer  and  Neuberg  ^),  and 
it  is  believed  that  the  urine  contains  a  skatol-chromogen  from  which  red  and 
^eddish-^•iolet  coloring-matters  are  obtained  by  decomposition  with  strong 
acids  and  an  oxidizing  agent. 

Skatoxyl-sulphuric  acid  originates,  if  it  exists  in  the  urine,  from  skatol 
w^hich  is  formed  by  putrefaction  in  the  intestine,  and  wliich  is  then  conju- 
gated with  sulphuric  acid  after  oxidation  into  skatoxyl.  That  skatol 
introduced  into  the  body  passes  partly  as  an  ethereal-sulphuric  acid  into 
the  urine  has  been  shown  by  Brieger.  Indol  and  skatol  act  differently,  at 
least  in  dogs;  indol  producing  a  considerable  amount  of  ethereal-sulphuric 

•  lirauss,  Zeitsclir.  f.  physiol.  Chem.,  IS;  Adrian,  ibid.,  19;  Wang,  ihid.,  25;  Sal- 
kowski,  ibid.,  42. 

^  Bouma,  Zeitsclir.  f.  physiol.  Chem.,  32;   Oerum,  ibid.,  43. 

3  Schmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  14;  G.  Hoppe-Seyler,  Zeitschr.  f. 
physiol.  Cliem.,  "  and  S;   Porcher  and  Her^-ieux,  Journ.  de  Physiol.,  7. 

■'Grober,  Miinch.  med.  Wochenschr. ,  1904;    Wang.  Salkowski's  Festschrift,  1904. 

*Otto,  Piiuger's  Arch.,  33;   Mayer  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  29. 


596  URINE. 

acid,  while  skatol  gives  only  a  small  quantity  (IMester^).  The  state- 
ments are  somewhat  contradictory  on  this  subject  and  the  behavior  is 
somewhat  unsettled.  According  to  Staal  the  chromogen  of  the  skatoj 
red  is  neither  a  conjugated  sulphuric  acid  nor  a  conjugated  glucuronic  acid. 

The  potassium  salt  of  skatoxyl-sulphuric  acid  is  crystalline;  it  dissolves 
in  water,  but  with  difficulty  in  alcohol.  A  watery  solution  becomes  deep 
violet  with  ferric  chloride,  and  red  with  concentrated  nitric  acid.  The  salt 
is  decomposed  by  concentrated  hydrochloric  acid  with  the  separation  of  a 
red  precipitate.  The  nature  of  this  red  coloring-matter  produced  by  the 
decomposition  of  skatoxyl-sulphuric  acid  is  not  well  known;  neither  has  the 
relationship  existing  between  this  and  other  red  coloring-matters  in  the 
urine  been  decided.  On  distillation  with  zinc-dust  the  skatol-chromogen 
yields  skatol. 

Urines  containing  skatoxyl  are  colored  dark  red  to  violet  by  Jaffe's  indi- 
can  test  even  on  the  addition  of  hydrochloric  acid  alone ;  with  nitric  acid  they 
are  colored  cherry -red,  and  red  on  warming  with  ferric  chloride  and  hydro- 
chloric acid.  The  coloring-matter  which  yields  skatol  with  zinc-dust  may 
be  removed  from  the  urine  by  ether.  Urines  rich  in  skatoxyl  darken  from 
the  surface  downward  when  allowed  to  stand  in  the  air,  and  may  become 
reddish,  violet,  or  nearly  black.  Rosin  is  of  the  opinion  that  no  skatol- 
chromogen  exists  in  human  urine,  and  that  the  observations  made  hereto- 
fore were  due  to  a  confusion  with  indigo  red  or  urorosein.  It  cannot  be 
disputed  that  derivatives  of  skatol  occur  in  the  urine,  while  the  recent  in- 
vestigations of  Staal,  Grosser,  Porcher,  and  Hervieux  -  indicate  that 
skatol-redand  urorose  in  are  identical  or  at  least  closely  related  pigments. 
Only  the  formation  of  skatol  by  distillation  with  zinc  powder  can  be  con- 
sidered as  a  positive  proof  as  to  the  skatol  nature  of  a  pigment. 

Salkowski  ^  has  demonstrated  that  the  occurrence  of  skatol-cnrhoxylic  acid 
(indol  acetic  acid),  CgHg.N.COOH,  in  normal  urine  is  probable.  This  is  also  a 
product  of  jnitref action.  When  introduced  into  the  animal  body  this  acid  re- 
appears unchanged  in  the  urine.  With  hydrochloric  acid  and  very  dilute  ferric- 
chloride  solution  it  gives  an  intense  violet  color  to  the  solution.  This  test 
responds  with  a  watery  solution  containing  1 :  10  000  of  skatol-carboxylic  acid. 

Aromatic  Oxyacids.  In  the  putrefaction  of  proteins  in  the  intestine, 
'paraoxyphemil-acetic  acid,  C6H4(OH).CH2COOH,  and  yaraoxyphenyl-'pro- 
-pionic  acid,  C6H4(OH).C2H4.COOH,  are  formed  from  tyrosine  as  an  inter- 
mediate step,  and  these  in  great  part  pass  unchanged  into  the  urine.  The 
quantity  of  these  acids  is  usually  very  small.     They  are  increased  under  the 

'  Brieger,  Bar.  d.  deutsch.  chem.  Gesellsch.,  12,  and  Zeitschr.  f.  physiol.  Chem., 
4,  414;    Mester,  ibid.,  12. 

*  Rosin,  Virchow's  Arch.,  123;  Staal,  Zeitschr.  f.  i^liysiol.  Chem.,  4G;  Grosser, 
ibid.,  44;   Porcher  and  Hendeux,  Compt.  rend.,  138,  and  Journ.  de  Physiol.,  7. 

3  Zeitschr.  f.  physiol.  Cliem.,  9. 


P-OXYPHEXYLACETIC   AND    HOMOGENTISIC  ACIDS.  597 

same  conditions  as  the  phenols,  especially  in  acute  phosphorus  poisoning, 
in  which  the  increase  is  considerable.  A  small  portion  of  these  oxyacids 
is  combined  with  sulphuric  acid. 

Besides  these  two  oxyacids  wliich  regularly  occur  in  human  urine  we 
sometimes  have  other  oxyacids  in  urines.  To  these  belong  homogentisic 
acid  and  uroleucic  acid,  the  first  of  which  forms  the  specific  constituents  of 
the  urine  in  most  cases  of  alcaptonuria,  oxymandelic  acid,  found  by  Schultzex 
and  E.IESS  in  urine  in  acute  atrophy  of  the  liver,  oxyhydroparacoumaric  acid, 
found  by  Blenderm.\nn  in  the  urine  on  feeding  rabbits  with  tyrosine,  gallic 
acid,  which,  according  to  Bau^l\nx,i  sometimes  appears  in  horse's  urine, 
and  kynurenic  acid  (oxyquinoUncarboxylic  acid),  which  up  to  the  present 
time  has  been  found  only  in  dog's  urine.  Although  all  these  acids  do  not 
belong  to  the  physiological  constituents  of  the  urine,  still  they  will  be 
treated  in  connection  with  these. 

Paraoxyphenylacetic   A ci d,  CgHgOs  =  CeH^ < ^^  ^^qqjj    ^^^ 

p-Oxyphenylpropionic     Acid     (Hydroparacoumaric      Acid),      C9Hio03  = 

C6H4<^jj^^jj^QQQjj,  are  crystalline  and  are  soluble  both  in  water  and 

in  ether.  The  one  melts  at  148°  C.  and  the  other  at  125°  C.  Both  give 
a  beautiful  red  coloration  on  being  warmed  with  Millox's  reagent. 

To  detect  the  presence  of  these  oxyacids  proceed  in  the  following  way  (Bau- 
MAXX):  Warm  the  urine  for  a  while  on  the  water-bath  with  hydrochloric  acid 
in  order  to  drive  off  the  volatile  phenols.  After  cooling  shake  three  times  with 
ether,  and  then  shake  the  ethereal  extracts  with  dilute  soda  solution,  which  dis- 
solves the  ox3-acids,  while  the  residue  of  the  phenols  which  are  soluble  in  ether 
remains.  The  alkaline  solution  of  the  oxyacids  is  now  faintly  acidified  with  sul- 
phuric acid,  shaken  again  with  ether,  the  ether  removed  and  allowed  to  evaporated 
the  residue  dissolved  in  a  little  water,  and  the  solution  tested  with  Millox's, 
reagent.  The  two  oxyacids  are  best  differentiated  by  their  different  melting- 
points.  The  reader  is  referred  to  other  works  for  the  method  of  isolating  and 
separating  these  two  oxyacids. 

Homogentisic   Acid  (Dioxyphenylacetic  Acid),  C8H804= 
/0H(1) 
CeHss— OH  (4)  .     This  acid,  which  was  discovered  by  Marshall  ^  and 

^CHo.COOHCo) 
by  him  called  glycosuric  acid,  was  isolated  in  larger  quantities  by  Wolkow 
and  BAU^L\xx  in  a  case  of  alcaptonuria  and  carefully  studied  by  them. 
They  called  it  homogentisic  acid  because  it  is  a  homologue  of  gentisic  acid, 
and  they  showed  that  the  peculiar  properties  of  so-called  alcaptonuric  urine 
in  this  case  were  due  to  this  acid.     This  acid  has  later  been  found  in  mam- 

*  Schultzen  and  Riess,  Chem.  Centralbl.,  1869;    Blendermann,  Zeitschr.  f.  phjrsiol. 
Chem.,  6,  267;    Baumann,  ibid.,  G,  193. 

*  The  Medical  News,  Philadelphia,  January  8,  1887. 


598  URINE. 

cases  of  alcaptonuria  by  Embden,  Garnier  and  Voirin,  Ogden,  Garrod, 
and  many  others.  Glycosuric  acid,  isolated  from  alcaptonuric  urine  by 
Geyger/  seems  to  be  identical  with  homogentisic  acid. 

The  quantity  of  acid  eliminated  is  increased  by  food  rich  in  protein.  On 
the  ingestion  of  tyrosine  by  persons  with  alcaptonuria,  Wolkow  and 
Baumann  and  Embden  observed  a  greater  quantity  of  homogentisic  acid 
in  the  urine.  Since  Langstein  and  E.  Meyer  showed  in  a  case  of  alcap- 
tonuria that  the  quantity  of  tyrosine  in  the  protein,  even  when  calculated 
to  a  maximum,  was  not  sufficient  to  account  for  the  quantity  of  homo- 
gentisic acid,  and  that  therefore  we  must  admit  of  another  source  (the 
phenylalanine)  for  the  alcapton,  Falta  and  Langstein  -  have  given  a 
direct  proof  that  homogentisic  acid  can  also  be  formed  from  phenylalanine. 
Tyrosine  and  phenylalanine  are  quantitatively  converted  into  homogentisic 
acid  in  alcaptonuria  (Falta).  Dil^romtyrosine,  on  the  contrary,  yields 
as  little  homogentisic  acid  as  bromine  or  iodine  derivatives  of  protein 
bodies  (Falta).  According  to  the  investigations  of  Langstein  and  AIeyer, 
and  especially  of  Falta,  different  proteins  yield  varying  quantities  of 
homogentisic  acid  in  alcaptonuria,  and  accordingly  larger  amounts  in  pro- 
portion as  the  protein  is  rich  in  tyrosine  and  phenylalanine. 

Wolkow^  and  Baumann  explain  the  formation  of  homogentisic  acid 
from  tyrosine  by  an  aljnormal  fermentation  in  the  upper  parts  of  the  in- 
testine, but  this  view  has  now  been  generally  rejected.  Homogentisic  acid 
is  burnt  in  the  healthy  organism,  and  in  consonance  with  the  views  of  Falta 
and  Langstein  alcaptonuria  is  considered  as  an  anomaty  in  the  metabolism. 
O.  Neubauer  and  Falta  ^  found  in  experiments  with  different  aromatic 
substances  that  the  aromatic  a-oxyacids  as  well  as  the  a-amino  acids  de- 
rived from  the  protein  bodies,  are  converted  into  homogentisic  acid  in  the 
organism  of  alcaptonurics.  It  can  be  admitted  with  Falta  that  the  phenyl- 
alanine in  the  body  by  deamidation  is  converted  into  phenyl-a-lactic  acid, 
C6H5.CH2.CHOH.COOH,  from  which  by  taking  up  two  hydroxyl  groups 
dioxyphenyl-a-lactic  acid  (uroleucic  acid),  (OH)2C6H3-CH2.CHOH.COOH, 
is  formed,  and  then  from  this  by  oxidation  dioxyphenylacetic  acid  (homo- 
gentisic acid),  (OH)2C6H3.CH2.COOH,  is  produced.  Tyrosine  also  is  sup- 
posed to  undergo  an  analogous  transformation  whereby  a  removal  of  OH 
groups  in  the  para  position  must  be  admitted  and  in  both  cases  the  homo- 
gentisic acid  formed  is  under  normal  conditions  further  destroyed  by  a 

^  Wolkow  and  Baumann,  Zeitschr.  f.  physiol.  Cliem.,  15;  Embden,  ibid.,  17  and 
^8;  Garnier  and  Voirin,  Arch,  de  Physiol.  (5),  l;  Ogden,  Zeitschr.  f.  physiol.  Chem., 
20;   Geyger,  cited  from  Embden,  1   c,  18. 

^  Langstein  and  Meyer,  Deutsch.  Arch.  f.  klin.  Med.,  78;  Falta  and  Langstein,  Zeit- 
schr. f.  physiol.  Chem.,  37;  Falta,  Der  Eiweiss-Stoffwechsel  bei  der  Alkaptonurie, 
Habilitationsschrift,  Naumburg  a.  S.,  1904. 

'  Zeitschr.  f.  physiol.  Chem.,  42. 


HOMOGENTISTIC   ACID.  599 

rupture  of  the  benzene  ring.  The  deniohtion  of  the  tyrosine  and  the 
phenylalanine  according  to  this  view  takes  place  in  normal  organisms  by 
way  of  the  alcaptonic  acids,  and  the  metabolic  anomaly  in  alcaptonuria 
consists  in  that  the  demolition  stops  at  this  point  and  the  property  of  the 
organism  in  alcaptonuria  of  rupturing  the  benzene  ring  is  absent. 

Garrod/  who  has  observed  several  cases  of  alcaptonuria,  has  also  tabu- 
lated about  forty  cases  of  alcaptonuria  which  he  finds  in  the  literature. 
From  this  he  shows  that  the  anomaly  of  the  protein  metaljolism  occurs 
oftener  in  males  than  in  females,  and  also  that  blood  relationship  of  the 
parents  (first  cousins)  predisposes  to  alcaptonuria. 

On  fusing  homogentisic  acid  with  alkali  it  yields  gentisic  acid  (hydro- 
quinone-carboxylic  acid)  and  hydroquinone.  When  introduced  into  the 
intestine  of  the  dog  a  part  is  converted  into  toluhydroquinone,  which  is 
eliminated  in  the  form  of  an  ethereal  sulphuric  acid.  Homogentisic  acid 
has  also  been  prepared  synthetically  by  Baumaxx  and  Fraxkel,^  starting 
with  gentisic  aldehyde. 

Homogentisic  acid  crystallizes  with  1  mol.  of  water  in  large,  trans- 
parent prismatic  crystals,  wliich  become  non-transparent  at  the  tempera- 
ture of  the  room  with  the  loss  of  water  of  crystallization.  They  melt  at 
146.5-147°  C.  They  are-  soluble  in  water,  alcohol,  and  ether,  but  nearly 
insoluble  in  chloroform  and  benzene.  Homogentisic  acid  is  optically  in- 
active and  non-fermentable.  Its  watery  solution  has  the  properties  of  so- 
called  alcaptonuric  urine.  It  becomes  greenish  brown  from  the  surface 
downward  on  the  addition  of  very  little  caustic  soda  or  ammonia  with 
excess  of  oxygen,  and  on  shaking  it  quickly  becomes  dark  brown  or  black. 
It  reduces  alkaline  copper  solutions  with  even  slight  heat,  and  ammoniacal 
silver  solutions  immediately  in  the  cold.  It  does  not  reduce  alkaline  bis- 
muth solutions.  It  gives  a  lemon-colored  precijiitate  with  Millox's  reagent, 
which  becomes  light  brick-red  on  warming.  Ferric  chloride  gives  to  the 
solution  a  blue  color  which  soon  disappears.  On  boiling  with  concentrated 
ferric-chloride  solution  an  odor  of  quinone  develops.  With  benzoyl  chloride 
and  caustic  soda  in  the  presence  of  ammonia  Ave  obtain  the  amide  of  diben- 
zoylhomogentisic  acid,  which  melts  at  204°  C,  and  v.hich  can  be  used  in 
the  isolation  of  the  acid  from  the  urine,  and  also  for  its  detection  (Ortox  and 
Garrod).  Among  the  salts  of  this  acid  must  be  mentioned  the  lead  salt 
containins:  water  of  crvstallization  and  34.79  ]ier  cent  Pb.  This  salt  melts 
at  214-215°  C.  '  ^ 

In  order  to  prepare  the  acid,  heat  the  urine  to  1 -oiling,  add  5  grams  of 
lead  acetate  for  everv  100  c.c,  filter  as  soon  as  the  lead  acetate  has  dis- 
solved, and  allow  the  filtrate  to  stand  in  a  cool  place  for  twenty -four  hours 
until  it  crystallizes  (Garrod).  The  dried,  powdered  lead  salt  is  suspended 
in  ether  and  decomposed  l)y  H2S.     After  the  spontaneous  evaporation   of 


'  Med.  chinirg.  Transact.,  1899  (where  all  known  cases  are  tabulated);    also   The 
Lancet,  1901  and  1902;   Garrod  and  Hele,  Journ.  of  Physiol.,  33. 
2  Zeitschr.  f.  phyaol.  Chem.,  20. 


600  URINE. 

the  ether  the  acid  is  obtained  in  nearly  colorless  crystals   (Orton    and 
Garrod  ^). 

In  regard  to  the  quantitative  estimation  we  proceed  according  to  the  sug- 
gestion of  Baumann  by  titrating  the  acid  with  a  N/10  silver  solution.  As  regards 
details  of  this  method  the  reader  is  referred  to  the  works  of  Baumann,  C.  Th. 
MoRNER  and  Mittelbach,  Garrod  and  Hurtley.  Deniges^  has  suggested 
another  method. 

Uroleucic  acid,  C9H10O5,  is,  according  to  Huppert,  probably  a  dioxyphenyl- 
lactic  acid,  CB,  (OH)2.CH2.CH(OH).COOH._  This  acid  was  first  prepared  by  Kirk 
from  the  urine  of  children  with  alcaptonuria,  which  also  contained  homogentisic 
acid.  Langstein  and  Meyer  ^  have  also  found  a  small  amount  of  this  acid  in 
a  case  of  alcaptonuria  studied  by  them.  It  melts  at  130-133°  C.  Otherwise, 
in  regard  to  its  behavior  with  alkalies,  with  access  of  air,  and  also  with  alkaline 
copper  solutions  and  ammoniacal  silver  solutions,  and  also  Millon's  reagent, 
it  is  similar  to  homogentisic  acid. 

Oxymadelic  acid,  CHsO^,  paraoxyphenylglycolic  acid,  HO.C„H,.CH(OH)COOH, 
is,  as  above  stated,  found  in  the  urine  in  acute  atrophy  of  the  liver.  The  acid 
crystallizes  in  silky  needles.  It  melts  at  162°  C,  dissolves  readily  in  hot  water, 
less  in  cold  water,  and  readily  in  alcohol  and  ether,  but  not  in  hot  benzene. 
It  is  precipitated  by  basic  lead  acetate,  but  not  by  lead  acetate. 

CH     COH 


HC      C      C.COOH, 
Kynurenic  acid  (;--oxy-^-quinolincarboxyhc  acid),  CicH7N03=     I       ||       | 

HC      C      CH 

CH  N 
has  only  been  found  thus  far  in  dogs'  urine;  its  quantity  is  increased  by  meat 
feeding.  According  to  the  observations  of  Glaessner  and  Langstein,  the  mother- 
substance  seems  to  be  contained  among  the  products  of  pancreatic  digestion 
which  are  soluble  in  alcohol  and  precipitable  by  acetone.  Ellinger  ■*  has 
recently  been  able  to  show  positively  that  tryptophane  is  the  mother-substance 
of  this  acid.  By  the  introduction  of  tryptophane  in  the  organism  he  has  shown 
the  formation  of  a  kynurenic  acid  not  only  in  dogs  but  also  in  ral)bits.  The 
acid  is  crystalline,  does  not  dissolve  in  cold  water,  rather  well  in  hot  alcohol, 
and  yields  a  barium  salt  which  crystallizes  in  triangular,  colorless  plates.  On 
heating  it  melts  and  decomposes  into  CO,  and  kynurin.  On  evaporation  to  dry- 
ness on  the  water-bath  with  hydrochloric  acid  and  potassium  chlorate  a  reddish 
residue  is  obtained  which  becomes  first  brownish  green  and  then  emerald-green 
on  adding  ammonia  (Jaffe's  reaction  ^). 

'Orton  and  Garrod,  Journ.  of  Physiol.,  27;    Garrod,  ibid.,  23. 

^  Mittelbach,  Deutsch.  Arch.  f.  klin.  Med.,  71  (which  contains  the  work  of  Baumann 
and  Morner);  Garrod  and  Hurtley,  Journ.  of  Physiol.,  33;  Denig?s,  Chem.  Centralbl. 
1897,  1,  338. 

3  Huppert,  Zeitschr.  f.  physiol.  Chem.,  23;  Kirk,  Brit.  Med.  Journ.,  1886  and  1888; 
Langstein  and  Meyer,  1.  c. 

*  Glaessner  and  Langstein,  Hofmeister's  Beitrage,  1 ;  Ellinger,  Ber.  d.  d.  chem. 
Gesellsch.,  37,  1804,  and  Zeitschr.  f.  physiol.  Chem.,  43.  The  older  literature  on 
ky::urenic  acid  may  be  found  in  Josephsohn,  Beitrage  zur  Kenntnis  der  Kynurensaure 
ausscheidung  beim  Hunde,  Inaug.-Dissert.,  Konigsberg,  1898. 

'  Zeitschr.  f.  physiol.  Chem.,  7.  In  regard  to  kynurenic  acid,  see  also  Huppert- 
Neubauer,  10.  Aufl.,  and  Mendel  and  Jackson,  Amer.  Journ.  of  Physiol.,  2;  Mendel 
and  Schneider,  ibid.,  5;    Camps,  Zeitschr.  f.  physiol.  Chem.,  33. 


URINARY    PIGMENTS.     UROCHROME.  601 

Urinary  Pigments  and  Chromogens.  The  yellow  color  of  normal  urine 
depends  perhaps  upon  several  pigments,  but  in  greatest  part  upon  urochrome. 
Besides  this  the  urine  seems  to  contain  a  very  small  quantity  of  hoemato^ 
porphyrin  as  a  regular  constituent.  Uroerythrin  also  is  of  frequent  occur- 
rence in  normal  urine,  but  not  always.  Finally,  the  excreted  urine  when 
exposed  to  the  action  of  light  regularly  contains  a  yellow  pigment,  urobilin, 
which  is  derived  from  a  chromogen,  urobilinogen,  by  the  action  of  light 
(Saillet)  and  air  (Jaffe,  Disque,^)  and  others.  Besides  this  chromogen, 
urine  contains  various  other  bodies  from  which  coloring-matters  may  be 
produced  by  tlie  action  of  chemical  agents.  Humin  substances  (perhaps 
in  part  from  the  carbohydrates  of  the  urine)  may  l^e  formed  by  the  action 
of  acids  (v.  Udranszky)  without  regard  to  the  fact  that  such  substances 
may  sometimes  originate  from  the  reagents  used,  as  from  impure  amyl 
alcohol  (v.  Udranszky  ^).  To  these  humin  bodies  developed  by  the  action 
of  acid  in  normal  urine  when  exposed  to  the  air  must  be  added  the  urophain 
of  Heller,  the  various  uromelanins  and  other  bodies  described  by  different 
investigators  (Plosz,  Thudichum,  Schunk^).  Indigo-blue  (uroglaucin  of 
Heller,  urocyanin,  cyanurin,  and  other  coloring-matters  of  older  investi- 
gators ^)  is  split  off  from  the  indoxyl-sulphuric  acid  or  indoxyl-glucoronic 
acid.  Red  coloring-matter  may  be  formed  from  the  conjugated  indoxyl 
and  skatoxyl  acids,  and  urohodin  (Heller),  urorubin  (Plosz),  urohcematin 
(Harley),  and  perhaps  also  iirorosein  (Nencki  and  Sieber  ^)  probabh' 
have  such  an  origin. 

We  cannot  discuss  more  in  detail  the  different  coloring-matters  obtained 
as  decomposition  products  from  normal  urine.  Htematoporphyrin  has 
already  been  referred  to  in  a  previous  chapter  (VI)  and  will  best  be  de- 
scribed in  connection  with  the  pathological  pigments.  It  only  remains  to 
describe  urochrome,  urobilin,  and  uroerythrin. 

Urochrome  is  the  name  given  by  Garrod  to  the  yellow  pigment  of  the 
urine.  Thudichum  ^  had  previously  given  the  same  name  to  a  less  pure 
pigment  isolated  by  himself.  According  to  Garrod  urochrome  is  free  from 
iron,  but  contains  nitrogen.  It  stands,  it  seems,  in  close  relationship  to 
urobilin,  as  Garrod  has  obtained  a  urobilin-like  pigment  by  the  action  of 
impure  aldehyde  on  urochrome,  and  Riva  '^  claims  that  urobilin  yields  a 

» Jaffe,  Centralbl.  f.  d.  med.  Wisaensch.  1868  and  1869,  and  Virchow's  .^Lrch.,  47; 
Disque,  Zeitschr.  f.  physiol.  Chem.,  2;    Saillet,  Revue  de  medecine,  17,  1897. 

^  V.  Udranszky,  Zeitschr.  f.  physiol.  Chem.,  11,  12,  and  13. 

^  Plosz,  Zeitschr.  f.  physiol.  Chem.,  8;  Thudichum,  Brit.  Med.  Journ.,  201,  and 
Journ.  f.  prakt.  Chem.,  104;   Schunk,  cited  from  Huppert-Neubauer,  10.  Aufl.,  509. 

*  See  Huppert-Neubauer,  161. 

*  In  regard  to  this  and  other  red  pigments,  see  Huppert-Neubauer,  593  and  597 ; 
Nencki  and  Sieber,  Journ.  f.  prakt.  Chem.  (2),  26. 

'Garrod,  Proceed.  Roy.  Soc,  55;   Thudichum,  1.  c. 

'  Garrod,  Journ.  of  Physiol.,  21  and  29;    Riva,  cited  from  Huppert-Neubauer,  524. 


602  URINE. 

body  similar  to  urochrome  on  careful  oxidation  with  permanganate.  Accord- 
ing to  Garrod  urobilin  can  be  converted  into  urochrome  by  evaporating  its 
aqueous  solution  containing  some  ether  on  the  water-bath.  The  fact  that 
urochrome  can  be  transformed  into  urobilin  ])y  means  of  active  acetaldehyde 
may  be  used,  according  to  Garrod,  as  a  means  of  detecting  urochrome. 

Urochrome  is,  according  to  Garrod,  amorphous,  brown,  very  readily 
soluble  in  water  and  ordinary  alcohol,  but  less  soluble  in  absolute  alcohol. 
It  dissolves  Init  slightly  in  acetic  ether,  amyl  alcohol,  and  acetone,  while  it 
is  insoluble  in  ether,  chloroform,  and  benzene.  Urochrome  is  precipitated 
by  lead  acetate,  silver  nitrate,  mercuric  acetate,  phosphotungstic  and  phos- 
phomolybdic  acids.  On  saturating  the  urine  with  ammonium  sulphate  a 
great  part  of  the  urochrome  remains  in  solution.  It  does  not  show  any 
absorption-bands  and  does  not  fluoresce  after  the  addition  of  ammonia  and 
zinc  chloride.  Urochrome  is  very  readliy  decomposed,  with  the  formation 
of  brown  substances,  by  the  action  of  acids.  According  to  Klemperer,'- 
urochrome  contains  4.2  per  cent  nitrogen. 

Urochrome  can  be  prepared  according  to  a  rather  complicated  method 
which  is  Ijased  upon  the  fact  that  the  substance  remains  in  great  part  in 
solution  on  saturating  the  urine  with  ammonium  sulphate.  If  the  proper 
quantity  of  alcohol  is  added  to  the  filtrate,  a  clear,  yellow  alcoholic  layer 
forms  OR  the  salt  solution,  which  contains  the  urochrome  and  which  can 
be  used  for  the  further  preparation  of  the  latter  (see  Garrod,  1.  c.)- 
Klemperer,  on  the  contrary,  removes  the  pigment  from  the  urine  by 
means  of  animal  charcoal,  washes  it  with  water  to  remove  the  indican 
and  other  bodies,  and  then  extracts  with  alcohol  and  uses  this  alcoholic 
extract  for  the  further  purification  according  to  Garrod. 

The  urochrome  can  l)e  quantitatively  estimated,  according  to  Klem- 
perer, by  a  colorimetric  method,  using  a  solution  of  tnie  yellow  G.  If 
0.1  gram  of  this  dye  is  dissolved  in  1  liter  of  water  and  5  c.c.  of  this  solu- 
tion diluted  to  50  c.c.  with  water,  then  this  solution  has  the  same  color  and 
shade  as  a  0.1  per  cent  urochrome  solution.  The  urine  must  be  diluted 
with  water  until  it  has  the  same  depth  of  color.  The  comparison  is  per- 
formed in  vessels  with  parallel  walls. 

Urobilin  is  the  pigment  first  isolated  from  the  urine  by  Jaff^i.^  and 
which  is  characterized  by  its  strong  fluorescence  and  by  its  absorption- 
spectnim.  Various  investigators  have  prepared  from  the  urine  by  different 
methods  pigments  w'hich  differed  slightly •  from  each  other  but  behaved 
essentially  like  Jaff^i's  urobilin.  Thus  different  urobilins  have  been  sug- 
gested, such  as  normal,  febrile,  physiological,  and  pathological  urobilins.^ 

'■  Berlin,  klin.  Wochenschr.,  40. 

^  Centralbl.  f.  d.  med.  Wissensch.,  1868  and  1869,  and  Virchow's  Arch.,  47. 

^  See  MacMunn,  Proc.  Roy.  Soc,  31  and  35;  Ber.  d.  deutsch.  chem.  Gesellsch.,  14, 
and  Journ.  of  Physiol.,  6  and  10;  Bogomoloff,  Maly's  Jahresber.,  22;  Eichholz,  Journ. 
of  Physiol.,  14;   Ad.  Jolles,  Pfliiger's  Arch.,  61. 


UROBILIN.  603 

The  possibility  of  the  occurrence  of  different  urobiUns  in  the  urine  cannot 
be  denied;  but  as  urobiUn  is  a  readily  changeable  body  and  difficult  to 
purify  from  other  urinary  ])igments,  the  question  as  to  the  occurrence  of 
different  urobilins  must  still  he  considered  open.  According  to  Saillet  ^ 
no  urobilin  exists  originally  in  human  urine,  but  only  the  mother-substance 
of  the  same.  urob^Uinogen,  from  which  the  uroljilin  is  formed  in  the  excreted 
urine  by  the  influence  of  light. 

Urobilin-like  bodies,  so-called  urobilinoids,  have  been  prepared  from 
bile-pigments  as  well  as  blood-pigments,  and  indeed  by  oxidation  as  well  as 
reduction.  ^Ialy  obtained  his  hydrolnlirubin  by  the  reduction  of  bilirubin 
wdth  sodium  amalgam,  and  Disque  obtained  a  prodvict  which  is  still  more 
similar  to  urobilin,  while  Stokvis  prepared  by  the  oxidation  of  cholecyanin 
with  a  little  lead  peroxide  a  choletelin  which  acted  ver}^  much  like  urobilin. 
Hoppe-Seyler,  Le  Nobel,  Nencki  and  Sieber  have  obtained  urobilinoid 
bodies  by  the  reduction  of  ha^matin  and  ha:'matoporphyrin  with  tin  or  zinc 
and  hydrochloric  acid,  while  jMacjMunn  ^  obtained  by  the  oxidation  of 
heematin  with  hydrogen  peroxide  in  alcohol  containing  sulphuric  acid  a 
pigment  Vv'hich  seemed  to  be  identical  with  urinary  urobilin.  It  is  apparent 
that  all  these  urobilins  cannot  be  identical. 

Many  investigators  declare  that  urobilin  is  identical  with  hydrobilimbin, 
but  according  to  the  researches  of  Hopkins  and  Garrod  ^  this  view  is  not 
correct,  because,  irrespective  of  other  small  differences,  each  body  has 
an  essentially  distinct  composition.  Hydrobihrubin  contains  C  64.68, 
H  6.93,  N  9.22  (jMaly),  w^hile  urinary  urobilin,  on  the  contrary,  contains 
C  63.46,  H  7.67,  N  4.09  per  cent.  The  urobilin  from  faeces,  stercohilin, 
has  the  same  composition  as  urinar}^  urobilin  with  4.17  per  cent  nitrogen. 

Urinary  urobilin  mav  not  be  identical  with  hydrobihrubin,  but  this  does 
not  exclude  the  possibility  that  urobilin,  according  to  the  generally  ad- 
mitted view,  is  derived  from  bilirubin  (although  not  by  simple  reduction 
and  taking  up  water)  in  the  intestine.  Several  ph}'siological  as  well  as 
clinical  observations  ^  speak  for  this  view,  among  which  we  must  mention 
the  regular  appearance  in  the  intestinal  tract  of  stercobilin,  undoubtedly 
derived  from  the  bile-pigments  and  having  the  same  composition  as  urinary 
urobilin,  the  absence  of  urobilin  in  the  urine  of  new-born  infants  as  well  as 

*  Revue  de  m^decine,  17,  1897. 

^  Maly,  Ann.  d.  Chem.  u.  Pharm.,  163;  Disqu^,  Zeitschr.  f.  physiol.  Chem.,  2; 
Stolcvis,  Centralbl.  f.  d.  med.  Wissensch.,  1873,  211  and  449;  Hoppe-Seyfer,  Ber.  d. 
deutsch.  chem.  Gesellscli.,  7;  Le  Nobel,  Pfli'iger's  Arch.,  40;  Nencki  and  Sieber, 
Monatshefte  f.  Chem.,  9,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  24;  MacMunn,  Proc.  Roy. 
Soc,  31. 

^  Journ.  of  Physiol.,  22. 

*See  Fr.  Miiller,  Scliles.  Gesellsch.  f.  vateri.  Kultur,  1892;  D.  Gerhardt,  "Ueber 
Hydrobilimbin  und  seine  Bezieh.  zum  Ikterus"  (Inaug.-Diss.,  Berlin,  1889);  Beck, 
Wien.  klin.  Wochenschr.,  1895;    Harley,  Brit.  Med.  Journ.,  1896. 


604  URINE. 

on  the  complete  exclusion  of  bile  from  the  intestine,  and  also  the  increased 
eUmination  of  urobihn  with  strong  intestinal  putrefaction.  On  the  other 
hand  there  are  investigators  who,  basing  their  opinion  on  clinical  observa- 
tions, deny  the  intestinal  origin  of  urobilin  and  claim  that  the  urobilin  is 
derived  from  a  transformation  of  the  bilirubin  elsewhere  than  in  the  intes- 
tine, by  an  oxidation  of  the  bile-pigment  or  by  a  transformation  of  the 
blood-pigments.^  The  possibility  of  a  different  mode  of  formation  of  uri- 
nary urobihn  in  disease  is  not  to  be  denied;  but  there  is  no  doubt  that 
this  pigment  is  formed  from  the  bile-pigments  in  the  intestine  under  physio- 
logical conditions. 

The  quantity  of  urobilin  in  the  urine  under  physiological  conditions  is 
very  variable.  Saillet  found  30-130  milligrams  and  G.  Hoppe-Seyler 
80-140  milligrams  in  one  day's  urine. 

There  are  numerous  observations  on  the  elimination  of  urobilin  in 
disease,  especially  by  Jaff]&,  Disqu6,  Gerhardt,  G.  Hoppe-Seyler,^  and 
others.  The  quantity  is  increased  in  hemorrhage  and  in  such  diseases 
where  the  blood-corj^uscles  are  destroyed,  as  is  the  case  after  the  action  of 
certain  bood-poisons,  such  as  antifibrine  and  antipyrine.  It  is  also  in- 
creased in  fevers,  cardiac  diseases,  lead  colic,  atrophic  cirrhosis  of  the  liver, 
and  is  especially  abundant  in  so-called  urobilin  icterus. 

The  properties  of  urobilin  may  be  different,  depending  upon  the  method 
of  preparation  and  the  character  of  the  urine  used ;  therefore  only  the  most 
important  properties  will  be  given.  Urobilin  is  amorphous,  brown,  reddish 
brown,  red,  or  reddish  yellow,  depending  upon  the  method  of  preparation. 
It  dissolves  readily  in  alcohol,  amyl  alcohol,  and  chloroform,  but  less 
readily  in  ether  or  acetic  ether.  It  is  less  soluble  in  water,  but  the  solu- 
bility is  augmented  by  the  presence  of  neutral  salts.  It  may  be  com- 
pletely precipitated  from  the  urine  by  saturating  with  ammonium  sulphate, 
especially  after  the  addition  of  sulphuric  acid  (MiiHU^).  It  is  soluble  in 
alkalies,  and  is  precipitated  from  the  alkaline  solution  by  the  addition  of 
acid.  It  is  partly  dissolved  by  chloroform  from  an  acid  (watery-alcohohc) 
solution;  alkali  solutions  remove  the  urobilin  from  the  chloroform.  The 
neutral  or  faintly  alkaline  solutions  are  precipitated  by  certain  metallic 
salts  (zinc  and  lead),  but  not  by  others,  such  as  mercuric  sulphate.  Uro- 
bilin is  precipitated  from  the  urine  by  phosphotungstic  acid.  It  does  not 
give  Gmelin's  test  for  bile-pigments.     It  gives,  on  the  contrary,  a  reaction 

'  In  regard  to  the  various  theories  as  to  the  formation  of  urobilin,  see  Harley, 
Brit.  Med.  Journ.,  1896;  A.  Katz.,  Wien.  med.  Wochenschr.,  1891,  Nos.  28-32;  Grimm, 
Virchow's  Arch.,  132;   Zoja,  Conferenze  cliniche  italiane,  Ser.  la,  1. 

^  In  regard  to  the  Hterature  on  this  subject  we  refer  the  reader  to  D.  Gerhardt, 
"Ueber  HydrobiHrubin  und  seine  Beziehungen  zum  Ikterus"  (BerHn,  1889),  and 
also  G.  Hoppe-Seyler,  Virchow's  Arch.,  124. 

'  Journ.  de  Pharm.  et  Chim.,  1878,  cited  from  Maly's  Jahresber.,  8. 


UROBILIN.  605 

which  may  be  mistaken  for  the  biuret  test,  by  the  action  of  copper  sulphate 
and  alkah.i 

Neutral  alcoholic  urobilin  solutions  are  in  strong  concentration  brownish 
5^ellow,  in  great  dilution  yellow  or  rose-colored.  They  have  a  strong  green 
fluorescence.  The  acid  alcoholic  solutions  are  brown,  reddish  yellow,  or 
rose-red,  according  to  concentration.  They  are  not  fluorescent,  but  show  a 
faint  absorption-band,  y,  between  h  and  F,  which  borders  on  F,  or  in  greater 
concentration  extends  over  F.  The  alkaline  solutions  are  brownish  yellow, 
yellow,  or  (the  ammoniacal)  yellowish  green,  according  to  concentration. 
If  some  zinc-chloride  solution  is  added  to  an  ammoniacal  solution  of  the  pig- 
ment it  becomes  red  and  shows  a  beautiful  green  fluorescence.  This  solu- 
tion, as  also  that  made  alkaline  with  fixed  alkalies,  shows  a  darker  and  more 
sharply  defined  band,  d,  between  h  and  F,  almost  midway  between  E  and  F. 
If  a  sufficiently  concentrated  solution  of  urobilin  alkali  is  carefully  acidi- 
fied with  sulphuric  acid  it  becomes  cloudy  and  shows  a  second  band  exactly 
at  E  and  connected  with  7- by  a  shadow  (Garrod  and  Hopkins,  Saillet^). 

Urobihnogen  is  colorless  or  is  oni}^  slightly  colored.  Like  urobilin  it  is 
precipitated  from  the  urine  by  saturating  with  ammonium  sulphate.  Ac- 
cording to  Saillet  it  may  be  extracted  by  acetic  ether  from  urine  acidi- 
fied with  acetic  acid.  It  dissolves  also  in  chloroform,  ethyl  ether,  and  amyl- 
alcohol.  It  shows  no  absorption-bands  and  is  readily  converted  into 
urobilin  by  the  influence  of  sunlight  and  oxygen,  and,  according  to  Neu- 
BAUER  and  Bauer,3  gives  the  Ehrlich  reaction  with  dime  thy  lamiclo- 
benzaldehyde  and  hydrochloric  acid  (see  below). 

In  preparing  urobilin  from  normal  urine,  precipitate  the  urine  with 
basic  lead  acetate  (Jaffe),  wash  the  precipitate  with  water,  dry  at  the 
ordinary  temperature,  then  boil  it  with  alcohol,  and  decompose  it  when 
cold  with  alcohol  containing  sulphuric  acid.  The  filtered  alcoholic  solution 
is  diluted  with  water,  saturated  wHh  ammonia,  and  then  treated  with  zinc- 
chloride  solution.  This  new  precipitate  is  washed  free  from  chlorine  with 
water,  boiled  with  alcohol,  dried,  dissolved  in  ammonia,  and  this  solution 
precipitated  with  sugar  of  lead.  This  precipitate,  Avhich  is  washed  with 
water  and  boiled  with  alcohol,  is  decomposed  by  alcohol  containing  sul- 
phuric acid,  the  filtered  alcoholic  solution  is  mixed  wdth  J  vol.  chloroform, 
diluted  with  water,  and  shaken  repeatedly,  but  not  too  energetically.  The 
urobilin  is  taken  up  l^y  the  chloroform.  This  last  is  washed  once  or  twice 
with  a  little  water  and  then  distilled,  leaving  the  uroljilin.  The  pigment 
may  be  precipitated  directly  from  the  urine  rich  in  uroliilin  by  ammonia 
and  zinc  chloride,  and  the  precipitate  treated  as  above  described  (Jaffe). 

'  See  Salkowski,  Berlin,  klin.  Wochenschr.,  1897,  and  Stokvis,  Zeitschr.  f,  Biologie, 
34. 

'  Garrod  and  Hopkins,  Journ.  of  Physiol.,  20;   Saillet,  1.  c. 

'  Neubauer,  cited  from  Centralbl.  f.  Physiol.,  19,  145;  Bauer,  cited  from  Biochem. 
Centralbl..  4,  .350. 


006  URINE. 

The  method  suggested  by  IMehu  (precipitation  with  ammonium  sul- 
phate) has  been  modified  by  Garrod  and  Hopkins  in  that  they  first  re- 
move the  uric  acid  by  saturating  with  ammonium  chloride  and  then  satu- 
rating the  filtrate  with  ammonium  sulphate.  The  precipitated  urobilin  is 
thus  made  purer  than  by  saturating  with  the  sulphate  directly.  The 
urobilin  is  extracted  from  the  dried  precipitate  by  a  great  deal  of  water, 
rei^recipitated  by  ammonium  sulphate,  and  this  procedure  rej^eated  several 
times  if  necessar}'.  The  dried  precipitate  finally  obtained  is  dissolved  in 
absolute  alcohol.  In  regard  to  small  details,  and  to  a  second  method  sug- 
gested by  these  experimenters,  we  refer  to  the  original  work.^ 

Saillet  extracts  the  urobilinogen  from  the  urine  by  shaking  with  acetic 
ether,  using  a  kerosene-oil  light.^ 

The  color  of  the  acid  or  alkaline  solution,  the  beautiful  fluorescence  of 
the  ammoniacal  solution  treated  with  zinc  chloride,  and  the  absorption- 
bands  of  the  spectrum,  all  serve  as  means  of  detecting  urobilin.  In  fever- 
urines  the  urobilin  may  be  detected  directly  or  after  the  addition  of  ammo- 
nia and  zinc  chloride  l)y  its  spectrum.  It  may  also  sometimes  be  detected 
in  normal  urine,  either  directly  or  after  the  urine  has  stood  exposed  to  the 
air  until  the  chromogen  has  been  converted  into  urobilin.  If  it  cannot 
be  detected  by  means  of  the  spectroscope,  then  the  urine  may  be  treated 
with  a  mineral  acid  and  shaken  with  ether  or,  still  better,  with  amyl  alcohol. 
The  amyl-alcohol  solution  is,  either  directly  or  after  addition  of  a  strongly 
ammoniacal  alcoholic  solution  of  zinc  chloride,  tested  spectroscopically. 
According  to  Schlesingf:r  ^  it  can  be  readily  detected  if  the  urine  is  pre- 
cipitated by  an  equal  volume  of  a  10  per  cent  solution  of  zinc  acetate  in 
absolute  alcohol.  Disturbing  bodies  are  here  precipitated  and  the  filtrate 
gives  the  fluorescence  directly,  and  also  the  spectrum.  Grimbert'*  has 
given  another  comparatively  simple  method. 

In  the  quantitative  estimation  of  urobilin  we  proceed  as  follows,  accord- 
ing to  G.  Hoppe-Seyler:  ^  100  c.c.  of  the  urine  is  acidified  with  sulphuric 
acid  and  saturated  with  ammonium  sulphate.  The  precipitate  is  collected 
on  a  filter  after  some  time,  washed  with  a  saturated  solution  of  ammonium 
sulphate,  and  repeatedly  extracted  with  equal  parts  of  alcohol  and  chloro- 
form after  pressing.  The  filtered  solution  is  treated  with  water  in  a  sepa- 
ratory  funnel  until  the  chloroform  separates  well  and  becomes  clear.  The 
chloroform  solution  is  evanorated  on  the  water-bath  in  a  weighed  beaker, 
the  residue  dried  at  100°  C,  and  then  extracted  wdth  ether.  The  ethereal 
extract  is  filtered,  the  residue  on  the  filter  dissolved  in  alcohol,  and  trans- 
ferred to  the  beaker  and  evaporated,  then  dried  and  weighed.  According 
to  this  method  G.  Hoppe-Seyler  found  0.08-0.14  gram  of  urobiUn  in  one 
day's  urine  of  a  healthy  person,  or  an  average  of  0.123  gram. 

Urobilin  may  also  be  determined  spcctrophotometrically  according:  to  Fr. 
MtJLLER  or  Saillet.^     Saillet  found  that  the  limit  for  the  perceptil^ility  of 

'  Joiirn.  of  Physiol.,  20. 

^  In  regard  to  this  and  other  methods,  we  must  refer  the  reader  to  special  works. 

'Deutsch.  med.  Wochenschr.,  1903. 

<See  Cliem.  Centralbl,  1904,  1,  1023. 

5  Virchow's  Arch.,  124. 

'Fr.  Miiller,  see  Huppert-Neubauer,  861;   Saillet,  1.  c. 


UROERYTHYRIN.  607 

the  absorption-bands  of  an  acid-urobilin  solution  lies  in  a  concentration  of 
1  milligram  of  urobilin  in  22  c.c.  of  solution  when  the  thickness  of  the  layer  of 
fluid  is  15  mm.  In  a  quantitative  estimation  the  urobilin  solution  is  diluted  to 
this  limit  and  then  the  quantity  of  urobilin  calculated  from  the  extent  of  dilu- 
tion. The  freshly  voided  urine,  shielded  from  light,  is  acidified  with  acetic  acid, 
completely  extracted  in  kerosene-oil  light  with  acetic  ether,  and  the  dissolved 
urobilinogen  oxidized  to  urobilin  with  nitric  acid.  On  the  addition  of  ammonia 
and  shaking  with  water  the  urobilin  passes  into  the  watery  solution.  This  is 
acichfied  with  hydi'ochloric  acid  and  diluted  until  the  above  limit  is  reached. 

Uroerythrin  is  the  pigment  which  often  gives  the  l)eautiful  red  color  to 
the  urinars^  sediments  {sedimentum  lateritium).  It  also  frequently  occurs, 
although  onl}'  in  ven-  small  quantities,  dissolved  in  normal  urines.  The 
quantity  is  increased  after  great  muscular  activity,  after  profuse  perspira- 
tion, immoderate  eating,  or  partaking  of  alcoholic  drinks,  as  well  as  after 
digestive  disturbances,  fevers,  circulatoiy  disturbances  of  the  liver,  and  in 
many  other  pathological  conditions. 

Uroerv'thrin,  which  has  been  especially  studied  by  Zoja,  Riva,  and 
Gaerod,^  has  a  pink  color,  is  amorjjhous,  and  is  verv  quickly  destroyed  by 
light,  especially  when  in  solution.  The  best  solvent  is  amvl  alcohol;  acetic 
ether  is  not  so  good,  and  alcohol,  chloroform,  and  water  are  even  less  valu- 
able. The  yery  dilute  solutions  show  a  pink  color;  but  on  greater  con- 
centration they  become  reddish  orange  or  bright  red.  They  do  not  fluoresce 
either  directly  or  after  the  addition  of  an  ammoniacal  solution  of  zinc  chlo- 
ride; but  they  have  a  strong  absorption,  beginning  in  the  middle  between 
D  and  E  and  extending  to  about  F,  and  consisting  of  two  bands  which 
are  connected  by  a  shadow  between  E  and  h.  Concentrated  sulphuric  acid 
colors  a  uroer^-thrin  solution  a  beautiful  carmine-red;  hydrochloric  acid 
gives  a  pink  color.  Alkalies  make  its  solutions  grass-green,  and  often  a 
play  of  colors  from  pink  to  purple  and  blue  is  observed.  Porcher  and 
HER^^EUx2  claim  that  uroer^-thrin  is  a  skatol  pigment. 

In  preparing  uroerythrin  according  to  Garrod,  the  sediment  is  dissolved 
in  water  at  a  gentle  heat  and  satm'ated  with  ammonium  chloride,  which  pre- 
cipitates the  pigment  with  the  ammonium  urate.  This  is  purified  by  repeated 
solution  in  water  and  precipitation  with  ammonium  chloride  until  all  the  urobilin 
is  removed.  The  precipitate  is  finally  extracted  on  the  filter  in  the  dark  with 
warm  water,  filtered,  then  diluted  with  water,  any  ha'matoporph^Tin  remaining 
being  removed  by  shaking  with  chloroform :  the  precipitate  is  then  faintly  acidi- 
fied with  acetic  acid  and  shaken  with  chloroform,  which  takes  up  the  uroerythrin. 
The  chloroform  is  evaporated  in  the  dark  at  a  gentle  heat. 

Volatile  fatty  adds,  such  as  formic  acid,  acetic  acid,  and  perhaps  also  but^Tic 
acid,  occur  under  normal  conditions  in  human  urine  (v.  Jaksch),  also  in  that  of 


^  Zoja,  Arch.  ital.  di  clinica  med.,  1S9.3.  and  Centralbl.  f.  d.  med.  "Wissensch. ,  1892; 
Riva,  Gaz.  med.  di  Torino,  Anno  43,  cited  from  Maly's  Jahresber.,  24;  Garrod,  Journ. 
of  Physiol.,  17  and  21. 

^  Journ.  de  Physiol.,  7. 


608  URINE. 

dogs  and  herbivora  (Schotten).  The  acids  poorest  in  carbon,  such  as  formic 
acid  and  acetic  acid,  are  more  constant  in  the  body  than  those  richer  in  carbon, 
and  therefore  the  relatively  greater  part  of  these  pass  unchanged  into  the  urine 
(Schotten).  Normal  human  urine  contains  besides  these  bodies  others  which 
yield  acetic  acid  when  oxidized  by  potassium  dichromate  and  sulphuric  acid 
(v.  Jaksch).  The  quantity  of  volatile  fatty  acids  in  normal  urine  calculated  as  acetic 
acid  is,  according  to  v.  Jaksch,  O.OOS-0.009  gram  per  twenty-four  hours;  accord- 
ing to  V.  RoKiTANSKY,  0.054  gram;  and  according  to  Magnus-Levy,  0.060  gram. 
The  quantity  is  increased  by  exclusively  farinaceous  food  (Rokitansky),  in 
fever  and  in  certain  diseases,  while  in  others  it  is  diminished  (v.  Jaksch,  Rosen- 
feld).  Large  amounts  of  volatile  fatty  acids  are  produced  in  the  alkaline  fer- 
mentation of  the  urine,  and  the  quantity  is  6-15  times  as  large  as  in  normal  urine 
(Salkowski  ')•  Non-volatile  fatty  acick  have  been  detected  as  normal  con- 
stituents of  urine  by  K.  Morner  and  Hybbinette.^ 

Paralactic  Acid.  It  is  claimed  that  this  acid  occurs  in  the  urine  of  healthy 
persons  after  very  fatiguing  marches  (Colasanti  and  Moscatelli).  It  is  found 
in  larger  amounts  in  the  urine  in  acute  phosphorus-poisoning  or  acute  yellow 
atrophy  of  the  liver  (Schultzen  and  Riess),  and  especially  abundant  in  eclampsia 
(Zweifel).  According  to  the  investigations  of  Hoppe-Seyler,  Araki,  and  v. 
Terray  lactic  acid  passes  into  the  urine  as  soon  as  the  supply  of  oxygen  is  de- 
creased in  any  way,  and  this  probably  explains  the  occurrence  of  lactic  acid  in 
the  urine  after  epileptic  attacks  (Inouye  and  Saiki).  Minkowski  ^  has  shown 
that  lactic  acid  occui's  in  the  urine  in  large  quantities  on  the  extirpation  of  the 
liver  of  birds. 

Glycerophosphoric  acid  occurs  as  traces  in  the  urine,*  and  it  is  probably  a 
decomposition  product  of  lecithin.  The  occurrence  of  succinic  acid  in  normal 
urine  is  a  subject  of  discussion. 

Carbohydrates  and  Reducing  Substances  in  the  Urine.  The  occurrence 
of  dextrose  as  traces  in  normal  urine  is  highly  probable,  as  the  investigations 
of  Brucke,  Abeles,  and  v.  UdrAnszky  show.  The  last  investigator  has 
also  shown  the  habitual  occurrence  of  carbohydrates  in  the  urine,  and 
their  presence  has  been  positively  proved  by  the  investigations  of  Baumann 
and  Wedenski,  and  especially  by  Baisch.  Besides  dextrose  normal  urine 
contains,  according  to  Baisch,  another  not  well-studied  variety  of  sugar; 
according  to  Lemaire,  probably  isomaltose  is  present,  and  besides  this  a 
dextrin-like  carbohydrate  (animal  gum),  as  shown  by  Landwehr,  Weden- 
ski, and  Baisch.  The  quantity  of  carbohydrates  eliminated  under  normal 
conditions  in  the  twenty-four  hours'  urine  and  determined  by  the  benzoyla- 


*  v.  Jaksch,  Zeitschr.  f.  physiol.  Chem.,  10;  Schotten,  ibid.,  7;  Rokitansky,  Wien. 
med.  Jahrbuch,  1887;  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  13;  Magnus-Levy,  Sal- 
kowski's  Festschrift,  1904;    Rosenfeld,  Deutsch.  med.  Wochenschr.,  29. 

2  Skand.  Arch.  f.  Physiol.,  7. 

^  Colasanti  and  Moscatelli,  Moleschott's  Untersuch.,  14;  Schultzen  and  Reiss, 
Chem.  Centralbl.,  1869;  Zweifel,  Arch.  f.  Gynakol,  76;  Araki,  Zeitschr.  f.  physiol. 
Chem.,  15,  16,  17.  19.  See  also  Irisawa,  ibid.,  17;  v.  Terray,  Pfli'iger's  Arch.,  65; 
Schutz,  Zeitsclu'.  f.  physiol.  Chem.,  19;  li.ouye  and  Saiki,  ibid.,  37;  Minkowski,  Arch, 
f.  exp.  Path.  u.  Pharm.,  21  and  31. 

*  See  Pasqualis,  Maly's  Jaliresber.,  24. 


CONJUGATED  GLUCORONATES.  609 

tion  method;  which  is  perhaps  not  sufficiently  trustworthy,  varies  consid- 
erably between  1.5  and  5.09  grams. ^ 

The  precipitate  obtained  from  concentrated  urine  by  the  aid  of  alcohol  and 
whose  nitrogen  (colloidal  nitrogen  according  to  Salkowski)  in  normal  urine 
amounts  to  2.34-4.08  per  cent  of  the  total  nitrogen  and  in  pathological  urines  to 
8-9  per  cent,  and  in  a  case  of  acute  yellow  atrophy  of  the  liver  to  21.8  per  cent, 
contains,  according  to  Salkowski,- a  nitrogenous  carbohydrate  which  has  strong 
reducing  action  upon  alkaline  copper  solutions  after  cleavage  with  hydrochloric 
acid. 

Besides  traces  of  sugar  and  the  reducing  substances  previously  men- 
tioned, uric  acid  and  creatinine,  the  urine  contains  still  other  bodies  of  this 
character.  These  latter  are  partly  conjugated  compounds  of  glucuronic 
acid,  CeHioOy,  which  is  closely  allied  to  dextrose.  The  reducing  power  of 
normal  urine  corresponds,  according  to  various  investigators,  to  1.5-5.96 
p.  m.  dextrose.^  That  portion  of  the  reduction  belonging  to  dextrose 
alone  is  equal  to  0.1-0.6  p.  m. 

Several  new  methods  for  the  determination  of  the  reducing  power  of  the  urine 
have  been  suggested.* 

Conjugated  glucoronates  occur,  as  indicated  by  Fluckiger  and  first 
positively  shown  by  Mayer  and  Neuberg,^  in  very  small  amounts  in  nor- 
mal urine.  They  occur  chiefly  as  phenol-  and  only  very  small  amounts 
of  indoxyl-  or  skatoxylglucuronates.  The  quantity  of  glucuronic  acid 
obtained  from  the  conjugated  glucuronates  is  estimated  as  0.04  p.  m.  by 
Mayer  and  Neuberg,  Besides  these  conjugated  glucuronates  perhaps 
sometimes  the  urine  contains  the  urea  glucuronic  acid,  the  ureidoglucu- 
ronic  acid  prepared  synthetically  by  Neuberg  and  Neimann.^ 

Very  large  amounts  of  these  conjugated  glucuronates  occur  in  the  urine, 
on  the  other  hand,  after  partaking  of  various  therapeutic  agents  and  other 
substances,  such  as  chloral  hydrate,  camphor,  naphthol,  borneol,  turpen- 
tine, morphine,  and  many  other  substances.  The  elimination  of  glucuronic 
acid  may  be  markedly  increased  in  severe  disturbances  of  the  respiration, 
severe  dyspnoea,  in  diabetes  mellitus,  and  by  the  direct  introduction  of  large 
amounts  of  dextrose.  According  to  P.  Mayer,  as  stated  on  page  122,  in  the 
oxidation  of  dextrose  a  part  of  it  forms  glucuronic  acid,  hence  it  is  to  be 

'  Lemaire,  Zeitschr.  f.  physiol.  Chem.,  21;  Baisch,  ibid.,  18,  19,  and  20.  In  these 
as  well  as  in  Treupel,  ibid.,  16,  the  works  of  other  investigators  are  cited.  See  also 
V.  Alfthan,  Deutsch.  med.  Wochenschr.,  26. 

^  Berlin,  klin.  Wochensclir.,  1905. 

^  Fliickiger,  Zeitschr.  f.  physiol.  Chem.,  9.     See  also  Huppert-Neubauer,  page  72. 

*  See  Rosin,  Miinch.  med.  Wochenschr.,  46;  Niemilowicz,  Zeitschr.  f.  physiol.  Chem., 
36;   Niemilowicz  with  Gittelmacher-Wilenko,  ibid.,  36,  and  Hdlier,  Compt.  rend.,  129. 

^  Fliickiger,  1.  c;   Mayer  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  29. 

•  Zeitschr.  f.  physiol.  Cliem.,  44. 


610  URINE. 

expected  that  the  glucuronic  acid  can  in  part  be  derived  from  the  dex- 
trose. As  a  conjugation  of  the  glucuronic  acid  with  other  bodies,  such 
as  aromatic  atomic  complexes,  prevents  the  combustion  of  this  acid  in 
the  animal  body,  it  ought  to  follow  that  after  the  introduction  of  such 
an  atomic  complex  in  the  body  during  a  glycosuria  a  corresponding 
reduction  of  the  glucose  elimination  would  take  place  with  the  increased 
excretion  of  conjugated  glucuronates.  In  order  to  prove  this  possibility 
O.  LoEWi  ^  fed  dogs  with  camphor  during  phlorhizin  diabetes  and  found 
that  the  above  expectation  was  not  realized.  Although  large  quantities  of 
campho-glucuronic  acid  were  excreted,  the  sugar  excretion  was  only  slightly 
diminished  and  not  in  proportion  to  the  quantity  of  conjugated  glucuronate 
excreted.  These  negative  results  are  contradicted  by  the  positive  results 
obtained  by  Paul  Mayer.^  Normally  rabbits  convert  nearly  all  the 
camphor  introduced  into  conjugated  glucuronic  acid.  According  to  Mayer 
if  we  allow  a  rabbit  to  starve  several  days  the  animal  becomes  so  poor 
in  the  mother-substance  (glycogen)  yielding  the  glucuronic  acid  that  the 
introduction  of  camphor  only  brings  about  an  elimination  of  small  quanti- 
ties of  glucuronic  acid.  By  the  simultaneous  administration  of  camphor 
and  dextrose  while  starvation  is  going  on,  the  elimination  of  glucuronic 
acid  rises  again  to  the  same  height  as  it  was  before  the  starvation  period. 
This  shows  that  the  sugar  had  conjugated  itself  with  the  camphor  as  glucu- 
r.onic  acid.  Hildebrandt  ^  has  also  made  experiments  showing  that  glucu- 
ronic acid  can  very  likely  be  formed  from  sugar.  The  observations  of 
M  ayer  are  not  substantiated  by  the  recent  investigations  of  Fenyvessy,* 
and  the  statements  on  this  question  are  contradictory. 

The  conjugated  glucuronic  acids  are  formed,  based  upon  the  investi- 
gations of  SuNDWiK,  Fischer  and  Piloty,5  by  a  combination  tagkin  place 
first  between  the  conjugator  and  the  dextrose  by  means  of  the  aldehyde 
group,  and  then  the  end  alcohol  group,  CH2OH,  is  oxidized  to  COOH.  The 
conjugated  glucuronic  acids  at  least  in  most  cases  seem  to  be  constructed 
after  the  glucoside  type,  a  view  which  has  received  further  support  by 
the  synthesis  of  phenolglucuronic  acid  and  euxanthonglucuronic  acids  by 
Neuberg  and  Neimann.6  Based  upon  their  cleavage  (as  far  as  they  have 
been  investigated)  by  kephir  lactase  and  emulsion,  but  not  by  yeast  lactase 
(Neuberg  and    Wohlgemuth '7),  the   conjugated  glucuronic   acids  must 

'  Arch.  f.  exp.  Path.  u.  Pharm.,  47. 
"Zeitschr.  f.  klin.  xMed.,  47. 
'"  Arch.  f.  exp.  Path.  u.  Pharm.,  44. 
*  See  Maly's  Jahresber.,  34. 

*E.    Sundwik,    Akademische    Abhandlung    Helsingfors,    1886;     see   also   Maly's 
Jahresber.,  10,  76;   Fischer  and  Piloty,  Ber.  d.  d.  chem.  Gesellsch.,  24. 
'  Zeitschr.  f.  physiol.  Chem.,  44. 
'  See  Neuberg,  Ergebnisse  der  Physiologic,  Bd.  3,  Abt.  1,  444. 


ORGANIC   COMBINATIONS  CONTAINING   SULPPIUR.  611 

belong  to  the  ,i9-series  of  glucosides.  The  ureidogkicuronic  acid  is  still  not 
constructed  upon  the  glucoside  type,  but  according  to  the  aldehydimine 
type,  H,N.C0.N.CH.(CH0H)4C00H.  The  reducing  urochloralic  acid  can 
hardly  be  built  upon  the  glucoside  type. 

According  to  the  body  with  which  they  are  conjugated  the  glucuronates 
show  different  behavior;  they  all  rotate  the  plane  of  polarization  to  the 
left,  while  the  glucuronic  acid  itself  is  dextrorotatory.  On  taking  up 
water  they  spUt  into  glucuronic  acid  and  the  conjugated  group.  They  are 
precipitated  by  basic  lead  acetate  or  by  basic  lead  acetate  and  ammonia. 
Most  of  the  conjugated  glucuronic  acids  do  not  have  a  reducing  action.  A 
few  reduce  copper  oxide  and  certain  other  metallic  oxides  in  alkahne  solu- 
tion and  hence  cause  errors  in  the  investigation  of  the  urine  for  sugar.  As 
the  detection  of  conjugated  glucuronic  acids  is  connected  with  the  tests  for 
sugar  in  the  urine,  we  will  treat  of  this  in  connection  with  these  tests. 

Organic  combinations  containing  sulphur  of  unknown  kind,  which  may 
in  small  part  consist  of  sulphocyanides,  0.04  (Gscheidlen)-O.II  p.  m. 
(I.  MuNK  1),  cystine  or  bodies  related  to  it,  taurine  derivatives,  chrondroitin- 
sulphuric  acid  and  protein  bodies,  but  in  greater  part  are  made  up  of  antoxy- 
proteic  acid,  oxyproteic  acid,  alloxyproteic  acid,  and  uroferric  acid,  are  found 
in  human  as  well  as  in  animal  urines.  The  sulphur  of  these  mostly  unknown 
combinations  has  been  called  "neutral,"  to  differentiate  it  from  the  "acid" 
sulphur  of  the  sulphate  and  ethereal-sulphuric  acids  (Salkowski  2) .  The 
neutral  sulphur  in  normal  urine  as  determined  by  Salkowski  is  15  per 
cent,  by  Stadthagen  13.3-14.5  per  cent,  and  by  Lepine  20  per  cent,  and 
Harnack  and  Kleine  ^  19-24  per  cent  of  the  total  sulphur.  In  starvation, 
according  to  Fr.  Mijller,  with  insufficient  supply  of  oxygen  (Reale  and 
Boeri,  Harnack  and  Kleine),  as  in  chloroform  narcosis  (Kast  and 
Mester),  as  also  after  the  introduction  of  sulphur  (Presch  and  Yvon^) 
the  quantity  of  neutral  sulphur  is  increased.  The  quantity  of  neutral 
sulphur  varies,  according  to  Benedict,  within  rather  narrow  limits  and 
especially,  according  to  Folin,  is  dependent  to  a  less  degree  than  the  sul- 
phate excretion  upon  the  extent  of  the  protein  metaboUsm.  The  relation- 
ship between  the  neutral  and  acid  sulphur  depends  in  the  first  place  upon 
the  extent  of  the  sulphuric-acid  excretion.  According  to  Harnack  and 
Kleine  5  the  relationship  of  the  oxidized  sulphur  to  the  total  sulphur 

'  Gscheidlen,  Pfli'iger's  Arch.,  14;   Munk,  Virchow's  Arch.,  69. 

^  Ibid.,  58,  and  Zeitsclir.  f.  physiol.  Chem.,  9. 

^Stadthagen,  Virchow's  Arch.,  100;    Lepine,  Compt.  rend.,  91  and  97;    Harnack 
and  Kleine,  Zeitschr.  f.  Biologic,  37. 

^  Fr.  Miiller,  Berl.  klin.  Wochenschr.,  1887;  Reale  and  Boeri,  Maly's  Jaliresber.,  24; 
Harnack  and  Kleine,  1.  c;  Presch,  Virchow's  Arch.,  119;  Yvon  Arch  de  Physiol 
(5),  10. 

^Benedict,  Zeitschr.  f.  klin.  Med.,  36;    Harnack    and  Kleine,  1.  c;    Folin,  Amer. 
Journ.  of  Physiol.,  13. 


612  URINE. 

changes  always  in  the  same  way  as  the  relationship  of  the  nitrogen  of  the 
urea  to  the  total  nitrogen.  The  more  unoxidized  sulphur  is  eliminated 
the  more  abundant  are  the  nitrogen  compounds,  not  urea,  in  the  urine — a 
Statement  which  coincides  with  recent  observations  showing  that  the 
neutral  sulphur  originates  chiefly  from  the  oxyproteic  acid,  the  alloxy- 
proteic  acid,  and  the  uroferric  acid. 

According  to  Lepine,  a  part  of  the  neutral  sulphur  is  more  readily  oxidized 
(directly  with  chlorine  or  bromine)  into  sulphuric  acid  than  the  other,  which  is 
only  converted  into  sulphuric  acid  after  fusing  with  potash  and  saltpeter.  Accord- 
ing to  the  investigations  of  W.  Smith,'  it  is  probable  that  the  most  unoxidizable 
part  of  the  neutral  sulphur  occurs  as  sulpho-acids.  An  increased  elimination  of 
neutral  sulphur  has  been  observed  in  various  diseases,  such  as  pneumonia,  cysti- 
nuria,  and  especially  where  the  flow  of  bile  into  the  intestine  is  prevented. 

The  total  quantity  of  sulphur  in  the  urine  is  determined  by  fusing  the  solid 
urinary  residue  with  saltpeter  and  caustic  alkali.  The  quantity  of  neutral  sulphur 
is  determined  as  the  difference  between  the  total  sulphur  and  the  sulphur  of  the 
sulphate  and  ethereal-sulphuric  acids.  The  readily  oxidiz  able  part  of  the  neutral 
sulphur  Is  determined  by  oxidation  with  bromine  or  potassium  chlorate  and 
hydrochloric  acid  (Lepine,  Jerome^). 

Sulphuretted  hydrogen  occurs  in  the  urine  only  under  abnormal  conditions 
or  as  a  decomposition  product.  This  compound  may  be  produced  from  the 
neutral  sulphur  of  the  organic  substances  of  the  urine  by  the  action  of  certain 
bacteria  (Fr.  Muller,  Salkowski  ').  Other  investigators  have  given  hypo- 
sulphites as  the  source  of  the  sulphuretted  hydrogen.  The  occurrence  of  hypo- 
sulphites in  normal  human  urine,  which  is  asserted  by  Heffter,  is  disputed  by 
Salkowski  and  Presch.^  Hyposulphites  occur  constantly  in  cat's  urine  and, 
as  a  rule,  also  in  dog's  urine. 

Antoxyjjroteic  acid  is  &  nitrogenous  SLCid  containing  sulphur  which  Bond- 
ZYNSKi,  DoMBROWSKi,  and  Panek  5  have  isolated  from  human  urine.  The 
composition  of  the  acid  was:  C  43.21,  H  4.91,  N  24.4.  S  0.61,  and  O 
26.33  per  cent.  A  part  of  the  sulphur  can  be  spUt  off  by  alkali.  This  acid 
is  soluble  in  water,  is  dextrorotatory,  and  is  precipitated  only  from  con- 
centrated solution  by  phosphotungstic  acid.  It  does  not  give  the  protein 
color  reactions,  but  gives  Ehrlich's  diazo-re action  (see  below).  The  salts 
with  the  alkalies,  barium,  calcium,  and  silver  are  soluble  in  water,  and  of 
these  salts  that  with  barium  and,  to  a  still  higher  degree,  the  silver  salt  are 
soluble  with  difficulty  in  alcohol.  The  free  acid  and  its  salts  are  precipi- 
tated by  mercuric  nitrate  and  acetate,  and  by  this  last  reagent  even  from 
solutions  strongly  acidified  wnth  acetic  acid.  Basic  lead  acetate  does  not 
precipitate  the  pure  acid. 

Oxyproteic  acid  is  the  name  given  by  Bondzinski  and  Gottlieb  ®  to  a 

»  L6pine,  1.  c;    Smith,  Zeitschr.  f.  physiol.  Chem.,  17. 

-  Jerome,  Pfliiger's  Arch.,  60. 

3Fr.  Miiller,  Berlin,  klin.  Woclienschr.,  1887;    Salkowski,  ibid.,  1888. 

*  Heffter,  Pfluger's  Arch.,  38;    Salkowski,  ibid.,  39;   Presch,  Virchow's  Arch.,  119. 

*  Zeitschr.  f.  physiol.  Chem.,  46. 

«  Centralbl.  f.  d.  med.  Wissensch.,  1897,  No.  33. 


ANTOXYPROTEIC  AND   ALLOXYPROTEIC   ACIDS.  613 

nitrogenous  acid  containing  sulphur  and  whicli  they  prepared  from  human 
urine,  which  has  recently  been  further  studied  by  Bondzynski,  Dom- 
BROWSKI  and  Panek.  This  acid  contained  C  39.62,  H  5.64,  N  18.08,  S  1.12, 
and  O  35.54  per  cent,  and  also  contains  sulphur  which  could  be  split  off. 
On  cleavage  it  yields  no  tyrosine,  nor  does  it  give  Ehrlich's  diazo  reaction, 
the  xanthoproteic  nor  the  biuret  reaction.  It  gives  a  faint  indication  of 
a  MiLLON  reaction  and  is  not  precipitated  by  phosphotungstic  acid,  hence 
it  leads  to  an  error  in  the  Pfluger-Bohland's  method  for  estimating  urea. 
The  acid  soluble  in  water  is  precipitated  by  mercuric  nitrate  and  acetate 
in  neutral  solutions,  but  is  not  precipitated  by  basic  lead  acetate.  The 
salts  of  this  acid  are  readily  soluble  in  water  and  more  soluble  in  alcohol 
than  the  corresponding  salts  of  antoxyproteic  acid. 

The  acid  which  is  found  in  large  quantities  especially  in  the  urine  of 
dogs  poisoned  with  phosphorus  (Bondzynski  and  Gottlieb)  is  considered 
like  the  preceding  acids  as  an  intermediary  oxidation  product  of  the  pro- 
teins, and  oxyproteic  acid  seems  to  represent  a  higher  state  of  oxidation  or 
a  demolition  of  the  proteins  than  the  antoxyproteic  acid. 

The  acid  called  uroproteic  acid  by  Cloetta  is  probably  a  mixture  of  several 
bodies,  according  to  the  recent  investigations  of  Bondzynski,  Dombrowski,  and 
Panek.  The  same  applies  also  to  the  barium  oxyproteate  prepared  by  Pregl  ' 
from  the  urine. 

AUoxy prottic  acid  is  a  third  acid  related  to  the  above,  which  was  first 
isolated  by  Bondzynski  and  Panek  ^  from  the  urine  and  then  carefully 
studied  with  Dombrowski.  The  composition  is:  C  41.33,  H  5.70,  N  13.55, 
S,  2.19,  and  0,  37.23  per  cent,  based  upon»new  investigations.  The  free 
acid  is  soluble  in  water.  It  gives  neither  the  biuret  reaction  nor  Ehrlich's 
reaction,  and  is  not  precipitated  by  phosphotungstic  acid.  Differing  from 
the  other  acids,  it  is  precipitated  by  basic  lead  acetate  and  its  salts  are  only 
slightly  soluble  in  alcohol. 

The  preparation  of  the  three  above-mentioned  acids  is  based  in  part 
upon  the  fact  that  alloxyproteic  acid  alone  is  precipitated  b}"  basic  lead 
acetate  and  that  the  two  other  acids  can  be  precipitated  from  the  filtrate 
by  mercuric  acetate,  the  antoxyproteic  acid  in  acetic  acid  solution,  and  the 
oxyproteic  acid  in  neutral  solution.  The  preparation  is  nevertheless  very 
tedious  and  complicated  and  therefore  we  must  refer  to  the  original  works  ^ 
for  details. 

Uroferric  acid  is  an  acid  isolated  by  Thiele  '^  from  the  urine,  according 
to   Siegfried's   method   for  preparing  pure   peptone.     It   also   contains 

'  Cloetta,  Arch.  f.  exp.  Path.  u.  Pharm.,  40;   Pregl,  Pfluger's  Arch.,  75. 
'  Ber.  d.  d.  chem.  Gesellsch.,  36. 
^  Zeitschr.  f.  physiol.  Chem.,  46. 
*Ibid.,  37. 


614  URINE. 

sulphur,  3.46  per  cent,  and  has  the  formula  CssHseNgSOig.  The  acid  forms 
a  white  powder  which  is  readily  soluble  in  water,  saturated  ammonium- 
sulphate  solution,  and  methji  alcohol.  It  is  soluble  with  difficulty  in  abso- 
lute alcohol,  insoluble  in  benzene,  chloroform,  ether,  and  acetic  ether. 
About  one  half  of  the  sulphur  can  be  split  off  as  sulphuric  acid  on  boiling 
with  hydrochloric  acid.  The  acid  gives  neither  the  biuret  test  nor  ^^Iillon's 
or  Adamkiewicz's  reactions.  It  is  precipitated  by  mercuric  nitrate  and 
sulphate,  and  also  by  phosphotungstic  acid.  This  acid  is  hexabasic  and  its 
specific  rotation  is  {a)o=  -32.5°.  On  cleavage  it  yields  melanine  sub- 
stances, sulphuric  acid,  aspartic  acid,  but  no  hexone  bases.  The  existence 
of  this  acid  is  disputed  by  Bondzynski,  Dombrowski  and  P.\nek. 

Abderhalden  and  Pregl  i  have  shown  that  human  urine  normally 
contains  compounds  which  stand  perhaps  in  close  relationship  to  the  poly- 
peptides and  which  on  hydrolysis  with  acids  yield  at  least  a  part  of  the 
moities  existing  in  the  protein  molecule.  In  the  case  investigated  they 
obtained  abundant  glycocoU,  also  leucine,  alanine,  glutamic  acid,  phenyL 
alanine,  and  probably  also  aspartic  acid.  The  relationship  between  these 
polypeptide-like  bodies  and  the  above-mentioned  proteic  acids  and  to  the 
uroferric  acid  has  not  been  investigated. 

Amino-acids  may,  when  they  are  introduced  in  large  amounts  into  the  body, 
also  pass  in  part  into  the  urine.  This  has  been  shown  for  r-alanine  by  R.  Hirsch 
for  the  dog,  and  by  Plaut  and  Reese  for  dog  and  man,  and  for  /--leucine  by 
Abderhaldex  and  Samuely  ^  in  rabbits.  Embden  and  Reese,  Forssner,  Abder- 
halden and  ScHiTTENHELM,  and  Samuely  ^  were  able,  by  means  of  the  naph- 
thaline sulphochloride  method,  to  detect  glycocoll  in  normal  human  urine,  and 
this  glycocoll  must  occur  in  the  urine  in  a  combination  which  is  readily  split 
by  alkali,  .\lthough  we  have  numerous  investigations,  other  amino-acids  besides 
glycocoll  could  not  be  detected  in  normal  human  urine,  while,  on  the  contrary, 
in  pathological  conditions  other  amino-acids  have  been  found  several  times. 
The  amino-acid  fraction  of  the  urine  seems  to  be  increased  in  starvation  and  in 
high  altitudes  (Loewy  ■*). 

Organic  combinations  containing  phosphorus  (glycerophosphoric  acid,  phospho- 
carnic  acid  (Rockwood),  etc.,  which  ^neld  phosphoric  acid  on  fusing  with  salt- 
peter and  caustic  alkali,  are  also  found  in  urine  (Lepine  and  Eitmonnet,  Oertel). 
With  a  total  elimination  of  about  2.0  grams  total  P0O5,  Oertel  found  on  an 
average  about  0.0.5  gram  PgOj  as  phosphorus  in  organic  combination.  According 
to  Symmers  5  the  organic  combined  phosphoric  acid  may  in  many  pathological 


'  Zeitschr.  f.  physiol.  Chem.,  46. 

'  R.  Hirsch,  Zeitschr.  f.  exp.  Path.  u.  Therap.,  1;  Plaut  and  Reese,  Hofmeister's 
Beitriige,  7;   Abderhalden  and  Samuely,  Zeitschr.  f.  physiol.  Chem.,  4". 

^  Embden  and  Reese,  Hofmeister's  Beitriige,  7 ;  G.  Forssner,  Zeitschr.  f .  physiol. 
Chem.,  47;   Abderhalden  and  Schittenlielm,  ibid.,  47;    Samuely,  ibid.,  47. 

*  Deutsch.  med.  Wochenschr.,  1905. 

^  Rockwood,  Arch.  f.  (Anat.  u.)  Physiol.,  1895;  Oertel.  Zeitschr.  f.  physiol.  Chem., 
26,  wliich  citas  the  other  Avorks.  See  also  Keller,  Zeitschr.  f.  pliysiol.  Cliem.,  29; 
Maridel  and  Oertel,  X.  Y.  Univ.  Bull.  Med.  Sciences,  i;  Symmers,  Journ.  of  Path,  and 
Bact.,  10. 


PTOMAINES  AND   LEUCOMAINES.  615 

conditions  be  25-50  per  cent  of  'he  total  phosphoric  acid.  In  lymphatic  leucaemia^ 
and  especially  in  degenerative  diseases  of  the  nervous  system,  the  quantity  may 
increase. 

Enzymes  of  various  kinds  have  been  isolated  from  the  urine.  Among  these 
may  be  mentioned  pejmn  (Brucke  and  others),  which,  according  to  ]\Iatthes, 
undoubtedly  originates  from  the  stomach,  and  a  diastatic  enzyme  (Cohnheim  and 
others)  and  trypsin^ 

Mucin.  The  nubecula  consists,  as  shown  by  K.  Morxer,^  of  a  mucoid  which 
contains  12.74  per  cent  N  and  2.3  per  cent  S.  This  mucoid,  which  apprarently 
originates  in  the  urinary  passages,  may  pass  to  a  slight  extent  into  solution  in 
the  urine.  In  regard  to  the  nature  of  the  mucins  and  nucleoalbumins  otherwise 
occurring  in  the  urine  we  refer  the  reader  to  the  pathological  constituents  of  the 
urine. 

Ptoviaines  and  leucomaines,  or  poisonous  substances  of  an  unknown  kind, 
which  are  often  described  as  alkaloidal  substances,  occur  in  normal  m-ine  (Pouchet, 
Bouchard,  Aducco,  and  others).  Under  pathological  conditions  the  quantity 
of  these  substances  may  be  increased  (Bouchard,  Lepine  and  Guerix,  Villiers, 
Griffiths,  Albu,  and  others).  Within  the  last  few  years  the  poisonous  proper- 
ties of  urine  have  been  the  subject  of  more  thorough  investigation,  especially  by 
Bouchard.  He  found  that  the  night  urine  is  less  poisonous  than  the  day  urine, 
and  that  the  poisonous  constituents  of  the  day  and  night  ui'ines  have  not  the 
same  action.  In  order  to  be  able  to  compare  the  toxic  power  of  the  urine  under 
different  conditions,  Bouch-VRD  determines  the  urotoxic  coefficiext,  which  is 
the  weight  of  rabbit  in  kilos  that  is  killed  by  the  quantity  of  m'ine  excreted  in 
twenty-four  hours  by  1  kUo  of  the  person  experimented  upon.^ 

Baumaxx  and  v.  Udraxszky  have  shown  that  ptomaines  may  occur  in  the 
urine  under  pathological  conditions.  Tliey  demonstrated  the  presence  of  the  two 
ptomaines  discovered  and  first  isolated  by  Brieger — putresciue,  C^KjjN,  (tetra- 
methylendiamine),  and  cadaverine,  CsHuX,  (pentamethylendianiine)— in  the  urine 
of  a  patient  suffering  from  cystinuria  and  catarrh  of  the  bladder.  Cadaverine 
has  later  been  fomid  by  Stadthagex  and  Brieger  in  the  luine  in  two  cases  of 
cystinuria.  Brieger,  v.  Udraxszky  and  Baumaxx,  and  Stadthagex  have 
shown  that  neither  these  nor  other  diamines  occur  under  physiological  condi- 
tions, while  DoMBROWSKi,  on  the  contrary,  found  cadaverine  besides  another 
ptomaine  with  the  formula  CjHuXOo  in  normal  urines,  and  Kutscher  and  Loh- 
MANN  *  have  found  neurine.  The  occurrence  in  normal  urine  of  any  "  urine 
poison  "  is  denied  by  certain  investigators,  such  as  Stadthagex,  Beck,  and  v.  d. 
Bergh.^  The  poisonous  action  of  the  urine,  according  to  them,  is  due  in  part 
to  the  potassium  salts  and  in  part  to  the  sum  of  the  toxicity  of  the  other  normal 
urinary  constituents  (urea,  creatinme,  etc.),  which  have  very  little  poisonous 
action  individually.  The  occurrence  of  special  urine  poisons  under  normal 
conditions  is  difficult  to  deny  on  account  of  numerous  statements  on  this  subject, 
although  the  chemical  laature  of  these  substances  is  still  not  sufficiently  known. 

Many  substances  have  been  observed  in  animal  urine  which  are  not  found  in 
human  ui'ine.     To  these  belong  the  above-described  kynurenic  acid,  urocanic  acid, 

'  In  regard  to  the  literature  on  enzymes  in  the  urine,  see  Huppert-Neubauer,  599 ; 
Matthes,  Arch.  f.  exp.  Path.  u.  Pharm.,  49. 

2  Skand.  Arch.  f.  Physiol.,  6. 

^  A  complete  bibliography  on  the  ptomaines  and  leucomaines  of  the  urine  is  found 
in  Huppert-Neubauer,  403. 

*  Baumann  and  Udranszky,  Zeitschr.  f.  physiol.  Chem.,  13;  Stadthagen  and  Brieger, 
Virchow's  Arch.,  115;  Dombrowski,  Arch,  polonais.  d.  sciences  bid.,  1903;  Kutscher 
and  Lohmann,  Zeitsclir.  f.  physiol.  Chem., '48. 

*  Stadthagen,  Zeitschr.  f.  kliu.  Med.,  15;  Beck,  Pfliiger's  Arch.,  71;  v.  d.  Bergh, 
Zeitschr.  f.  klin.  Med.,  35. 


616  URINE 

also  found  in  dog's  urine  and  which  seems  to  stand  in  some  relationship  to  the 
purine  bases;  damaluric  acid  and  damolic  acid  (according  to  Schotten,'  probably 
a  mixture  of  benzoic  acid  with  volatile  fatty  acids),  obtained  by  the  distillation 
of  cow's  urine;  and  lastly  lithuric  acid,  fomid  in  the  urinary  concrements  cf 
certain  animals. 

III.  Inorganic  Constituents  of  Urine. 

Chlorides.  The  chlorine  occurring  in  the  urine  is  undoubtedly  combined 
with  the  bases  contained  in  this  excretion;  the  chief  part  is  in  combination 
with  sodium.  In  accordance  with  this,  the  quantity  of  chlorine  in  the 
urine  is  generally  expressed  as  NaCl. 

The  question  as  to  whether  a  part  of  the  chlorine  contained  in  the  urine 
exists  as  organic  combinations,  as  considered  by  Berlioz  and  Lepinois,  is 
still  disputed  .2 

The  quantity  of  chlorine  combinations  in  the  urine  is  subject  to  con- 
siderable variation.  In  general  the  quantity  from  a  healthy  adult  on  a 
mixed  diet  is  10-15  grams  of  NaCl  per  twenty -four  hours.  The  quantity  of 
common  salt  in  the  urine  depends  chiefly  upon  the  quantity  of  salt  in  the 
food,  with  which  the  elimination  of  chlorine  increases  and  decreases.  The 
free  drinlving  of  water  also  increases  the  elimination  of  chlorine,  which  is 
greater  during  activity  than  during  rest  (at  night) .  Certain  organic  chlorine 
combinations,  such  as  chloroform,  may  increase  the  elimination  of  inorganic 
chlorides  by  the  urine  (Zeller,  Kast^). 

In  diarrhoea,  in  quick  formation  of  large  transudates  and  exudates,  also 
in  specially  marked  cases  of  acute  febrile  diseases  at  the  time  of  the  crisis, 
the  elimination  of  NaCl  is  materially  decreased.  The  excretion  of  chlorine 
may  vary  considerably  in  disease,  but  still  the  NaCl  taken  with  the  food 
has  here,  as  in  physiological  conditions,  a  great  influence  on  the  NaCl 
excretion.'* 

The  quantitative  estimation  of  chlorine  in  the  urine  is  most  simply  per- 
formed by  titration  with  silver-nitrate  solution.  The  urine  must  not  con- 
tain either  proteid  (w^hich  if  present  must  be  removed  by  coagulation)  or 
iodine  or  bromine  compounds. 

In  the  presence  of  bromides  or  iodides  evaporate  a  measured  quantity  of  the 
urine  to  dryness,  fuse  the  residue  with  saltpeter  and  soda,  dissolve  the  fused 

'  Zeitschr.  f.  physiol.  Chem.,  7. 

'Berlioz  and  Lepinois,  see  Chem.  Centralbl.,  1894,  1,  and  1895,  1;  also  Petit  and 
Terrat,  ibid.,  1894,  2,  and  Vitali,  ibid.,  1897,  2;  Viele  and  Moitessier,  Maly's  Jahresber., 
31;   Meillere,  ibid.;   Bruno,  ibid.,  452. 

^  Zeller,  Zeitschr.  f.  physiol.  Chem.,  8;  Kast,  ibid.,  11;  Vitali,  Chem.  Centralbl., 
1899,  2. 

*  On  the  elimination  of  chlorine  in  disease,  see  Albu  and  Neuberg,  Physiol,  u.  Pathol, 
des  Mineralstoffwechsels,  Berlin,  1906. 


CHLORIDES.  617 

mass  in  water,  and  remove  the  iodine  or  bromine  by  the  addition  of  dilute  sul- 
phuric acid  and  some  nitrite,  and  thoroughly  shake  with  carbon  disulphide. 
The  liquid  thus  obtained  may  now  be  titrated  with  silver  nitrate  according  to 
Volhard's  method.  The  quantity  of  bromide  or  iodide  is  calculated  as  the 
difference  between  the  quantity  of  silver-nitrate  solution  used  for  the  titration 
of  the  solution  of  the  fused  mass  and  the  quantity  used  for  the  corresponding 
volume  of  the  original  urine. 

The  othei'^N'ise  excellent  titration  method  of  ]\Iohr,  according  to  Avhich 
we  titrate  with  silver  nitrate  in  neutral  liquids,  using  neutral  potassium 
chromate  as  an  indicator,  cannot  be  used  directly  on  the  urine  in  careful 
work.  Organic  urinaiy  constituents  are  also  precipitated  by  the  silver  salt, 
and  the  results  are  therefore  somewhat  high  for  the  chlorine.  If  this  method 
is  to  be  employed,  the  organic  urinary  constituents  must  first  ]je  destroyed. 
For  this  purpose  evaporate  to  drs-ness  5-10  c.c.  of  the  urine,  after  the 
addition  of  1  gram  of  chlorine-free  soda  and  1-2  grams  chlorine-free  salt- 
peter, and  carefully  fuse.  The  mass  is  dissolved  in  water,  acidified  faintly 
wdth  nitric  acid,  and  then  neutralized  exactly  witli  pure  calcium  carbonate. 
This  neutral  solution  is  used  for  the  titration. 

The  silver-nitrate  solution  may  be  a  N/10  one.  It  is  often  made  of 
such  a  strength  that  each  cubic  centimeter  corresponds  to  0 .006  gram  CI  or 
0.01  gram  XaCl.  This  last-mentioned  solution  contains  29.075  grams  of 
AgNOs  in  1  liter. 

Freuxd  and  Toepfer,  as  well  as  Bodtker.^  have  suggested  modifica- 
tions of  Mohr's  method. 

Volhard's  ^Method.  Instead  of  the  preceding  determination,  Vol- 
hard's method,  which  can  be  performed  directly  on  the  urine,  maj^  be 
employed.  The  principle  is  as  follows:  All  the  chlorine  from  the  urine 
acidified  with  nitric  acid  is  precipitated  by  an  excess  of  silver  nitrate, 
filtered,  and  in  a  measured  part  of  the  filtrate  the  quantity  of  silver  added 
in  excess  is  determined  b}'  means  of  a  sulphocyanide  solution.  This  excess 
of  silver  is  completely  precipitated  by  the  sulphocyanide,  and  a  solution  of 
some  ferric  salt,  which,  as  is  well  known,  gives  a  blood-red  reaction  with 
the  smallest  quantity  of  sulphocyanide,  is  used  as  an  indicator. 

We  require  the  following  solutions  for  this  titration:  1.  A  silver- 
nitrate  solution  which  contains  29.075  grams  of  AgNOs  per  liter  and  of 
which  each  cubic  centimeter  corresponds  to  0.01  gram  NaCl  or  0.00607  gram 
CI.  2.  A  saturated  solution  at  the  ordinary^  temperature  of  chlorine-free 
iron  alum  or  ferric  sulphate,  3.  Chlorine-free  nitric  acid  of  a  specific 
graA^ty  of  1.2.  4.  A  potassium-sulphocyanide  solution  which  contains  8.3 
grams  KCNS  per  liter,  and  of  which  2  c.c.  corresponds  to  1  c.c.  of  the 
silver-nitrate  solution. 

About  9  grams  of  potassium  sulphocyanide  is  dissolved  in  water  and  diluted 
to  1  liter.  The  quantity  of  KCXS  contained  in  this  solution  is  determined  by  the 
silver-nitrate  solution  in  the  follo'ning  way:  Measure  exactly  10  c.c.  of  the  silver 
solution  and  treat  it  with  5  c.c.  of  nitric  acid  and  1-2  c.c.  of  the  ferric-salt  solu- 
tion and  dilute  vaih  water  to  about  100  c.c.  Now  the  sulphocyanide  solution 
is  added  from  a  burette,  constantly  stirring  until  a  permanent  faint-red  colora- 
tion of  the  liquid  takes  place.     The  quantity  of  sulphocyanide  fomad  in  the  solu- 

^Freund  and   Toepfer,  see  Maly's  Jahresber.,  22;    Bodtker,  Zeitschr.  f.  phyhiol 
Chem..  20. 


618  URINE. 

tion  by  this  means  indicates  how  much  it  must  be  diluted  to  be  of  the  proper 
strength.  Titrate  once  more  with  10  c.c.  of  AgNOg  solution  and  correct  the  sul- 
phocyanide  solution  by  the  careful  addition  of  water  until  20  c.c.  exactly  corre- 
sponds to  10  c.c.  of  the  silver  solution. 

The  determination  of  the  chlorine  in  the  urine  is  performed  by  this 
method  in  the  following  way  Exactly  10  c.c.  of  the  urine  is  placed  in  a 
flask  which  has  a  mark  corresponding  to  100  c.c.  and  which  is  provided 
with  a  stopper;  5  c.c.  of  nitric  acid  is  added;  dilute  with  about  50  c.c.  of 
water  and  then  allow  exactly  20  c.c.  of  the  silver-nitrate  solution  to  flow 
in.  Close  the  flask  with  the  stopper  and  shake  well,  remove  the  stopper 
and  wash  it  with  distilled  water  into  the  flask,  and  fill  the  flask  to  the 
100-c.c.  mark  with  distilled  water.  Close  again  with  the  stopper,  carefully 
mix  by  shaking,  and  filter  through  a  dry  filter.  Measure  ofT  50  c.c.  of 
the  filtrate  by  means  of  a  dry  pipette,  add  3  c.c.  of  ferric-salt  solution,  and 
allow  the  sulphocyanide  solution  to  flow  in  until  the  liquid  above  the 
precipitate  has  a  permanent  red  color.  The  calculation  is  very  simple. 
For  example,  if  4.6  c.c.  of  the  sulphocyanide  solution  was  necessary  to 
produce  the  final  reaction,  then  for  100  c.c.  of  the  filtrate  (=10  c.c.  urine) 
9.2  c.c.  of  this  solution  is  necessary.  9.2  c.c.  of  the  sulphocyanide  solution 
corresponds  to  4.6  c.c.  of  the  silver  solution,  and  since  20—4.6=15.4  c.c. 
of  the  silver  solution  was  necessary  to  completely  precipitate  the  chlorine 
in  10  c.c.  of  the  urine,  then  10  c.c.  contains  0.154  gram  of  NaCl.  The 
quantity  of  sodium  chloride  in  the  urine  is  therefore  1.54  ])er  cent,  or  15.4 
p.  m.  If  we  always  use  10  c.c.  for  the  determination,  and  always  20  c.c. 
of  AgNOs  solution,  and  dilute  with  water,  to  100  c.c,  the  quantity  of 
NaCl  in  1000  parts  of  the  urine  is  found  by  subtracting  from  20  the  number 
of  cubic  centimeters  of  sulphocyanide  (R)  required  with  50  c.c.  of  the 
filtrate.     The  quantity  of  NaCl  p.  m.  therefore  under  these  circumstances 

=  20— R,  and  the  percentage  of  NaCl  =  '"  . 

If  it  is  necessary  to  destroy  the  organic  urinary  constituents  before  titration, 
this  can  best  be  performed,  according  to  Dehn,'  by  evaporating  the  urine  (10  c.c), 
after  the  addition  of  a  small  amount  of  sodium  peroxide,  to  dryness  on  the  water- 
bath  then  faintly  acidifying  with  nitric  acid  and  then  titrating  according  to 
VoLHARD.     Incineration  is  umiecessary. 

For  the  approximate  estimation  of  chlorine  in  the  urine  Ekehorn  has 
made  use  of  Volhard's  titration  method  ])y  using  for  the  determination 
a  glass  tube  closed  at  one  end  and  divided  into  half  cubic-centimeters 
and  called  the  chlorometer.  The  reagents  necessary  are:  (a)  a  mixture 
of  20  c.c  silver-nitrate  solution  (according  to  Volhard),  5  c.c  nitric  acid 
and  water  to  100  c.c;  (b)  40  c.c.  sulphocyanide  solution  (according  to 
Volhard)  and  60  c.c.  of  a  ferric  alum,  chlorine-free  and  saturated  at  the 
ordinary  temperature.  The  silver-nitrate  solution,  of  which  each  cul)ic 
centimeter  corresponds  to  0.002  gm.  NaCl,  is  equivalent  to  the  iron  sulpho- 
cyanide solution.  First  2  c.c.  of  the  urine  is  placed  in  the  graduated  tube 
and  then  0.5  c.c.  sul])hocyanide  solution,  and  the  silver-nitrate  solution 
gradually  added  (shaking  the  tube  closed  with  a  rubber  stopper)  until 
the  coloration  of  the  sulphocyanide   just  disappears.      0.5  c.c  is   sub- 

^  Zcitschr.  f.  physiol.  Chcm.,  44. 


PHOSPHATES.  619 

tracted  from  the  silver  solution  for  the  0.5  c.c.  of  the  sulphocyanide;  the 
tube  is  so  graduated  that  the  quantity  of  NaCl  in  the  urine  in  parts  per 
thousand  is  read  off  directly  on  the  tube.  The  difference  between  these 
results  and  those  obtained  by  Volhard's  titration  method  amounts  only, 
according  to  C.  Th.  Morner/  to  0.25  to  at  most  0.5  p.  m. 

The  approximate  estimation  of  chlorine  in  the  urine  (which  must  be 
free  from  proteid)  is  made  by  strongly  acidifying  with  nitric  acid  and  then 
adding  to  it,  drop  by  drop,  a  concentrated  silver-nitrate  solution  (1:8). 
In  a  normal  quantity  of  chlorides  the  droj)  .sinks  to  the  bottom  as  a  rather 
compact  cheesy  lump.  In  diminished  c^uantities  of  chlorides  the  precipi- 
tate is  less  compact  and  coherent,  and  in  the  presence  of  very  little  chlorine 
a  fine  white  precipitate  or  only  a  cloudiness  or  opalescence  is  oljtained. 

Phosphates.  Phosphoric  acid  occurs  in  acid  urines  partly  as  dihydrogen, 
MH2PO4,  and  partly  as  monohydrogen  M2HPO4,  phosphates,  both  of 
which  may  be  found  in  acid  urines  at  the  same  time.  Ott  2  found  that  on 
an  average  60  per  cent  of  the  total  phosphoric  acid  was  di-  and  40  per 
cent  was  monohydrogen  phosphate.  The  total  quantity  of  phosphoric  acid 
is  very  variable  and  depends  on  the  character  and  the  quantity  of  food.  The 
average  quantity  of  P2O5  is  in  round  numbers  2.5  grams,  with  a  variation  of 
1-5  grams  per  day.  A  small  part  of  the  phosphoric  acid  of  the  urine  orig- 
inates from  the  burning  of  organic  compounds,  such  as  nuclein,  protagon, 
and  lecithin,  within  the  organism;  on  exclusive  feeding  with  substances  rich 
in  nuclein  or  pseudonuclein  the  quantity  of  phosphates  is  essentially  in- 
creased ;  still  it  is  undecided  to  what  extent  the  excretion  of  phosphoric  acid 
is  a  measure  of  the  absorption  and  decomposition  of  these  bodies.^  The 
greater  part  originates  from  the  phosphates  of  the  food ,  and  the  quantity  of 
phosphoric  acid  eliminated  is  greater  when  the  food  is  rich  in  alkali  phos- 
phates in  proportion  to  the  quantity  of  lime  and  magnesium  phosphates. 
If  the  food  contains  much  lime  and  magnesia,  large  quantities  of  earthy 
phosphates  are  eliminated  by  the  excrement;  and  even  though  the  food 
contains  considerable  amounts  of  phosphoric  acid  in  these  cases,  the  quan- 
tity excreted  by  the  urine  is  small.  This  is  true  especially  of  herbivora, 
in  which  the  kidneys  are  the  chief  organs  for  the  excretion  of  alkali  phos- 
phates. In  man,  according  to  Ehrstrom,  the  content  of  lime  in  the  food 
seems  to  play  no  important  role,  as  in  his  experiments  aljout  one-half  of 
the  phosphoric  acid  taken  as  CaHP04  was  absorbed;  still  the  extent  of 
phosphoric-acid  excretion  through  the  urine  depends  in  man  not  only  upon 
the  total  quantity  of  phosphoric  acid  in  the  food,  l;)ut  also  upon  the  relative 

'  Ekehorn,  Hygiea,  Stockholm,  1906;   Morner,  Upsala  Lakaref.  Forh.  (N.  F.),  11. 

^  Zeitschr.  f.  physiol.  Chem.,  10. 

^  See  A.  Gumlich,  Zeitschr.  f.  physiol.  Chem.,  18;  Roos,  ibid.,  21;  Weintraud, 
Arch.  f.  (Anat.  u.)  Physiol.,  1895;  Milroy  and  Malcolm,  Journ.  of  Physiol.,  23;  Roh- 
mann  and  Steinitz,  Pfliiger's  Arch.,  72;  Loewi,  Arch.  f.  exp.  Path.  u.  Pharm.,  41 
and  45. 


620  URINE. 

amounts  of  the  alkaline  earths  and  the  alkali  salts  of  the  food.  In  car- 
nivora,  in  which  phosphate  injected  subcutaneously  is  eliminated  by  the 
intestine  (Bergman x),  the  urine  is  habitually  poor  in  phosphates. ^ 

As  the  extent  of  the  elimination  of  phosphoric  acid  is  mostly  dependent 
upon  the  character  of  the  food  and  the  absorption  of  the  phosphates  in  the 
intestine,  it  is  apparent  that  the  relationship  between  the  nitrogen  and 
phosphoric-acid  excretion  cannot  run  parallel.  This  is  in  fact  so,  and, 
according  to  Ehrstrom,  the  organism  has  the  power  of  accumulating  large 
quantities  of  phosphorus  for  a  relatively  long  time  independent  of  the 
condition  of  the  nitrogen  balance.  With  a  certain  regular  food  the  rela- 
tionship between  nitrogen  and  phosphoric  acid  in  the  urine  can  be  kept 
nearly  constant.  Thus  on  feeding  with  an  exclusive  meat  diet,  as  ob- 
served by  Voit2  in  dogs,  when  the  nitrogen  and  phosphoric  acid  (P2O5) 
of  the  food  exactly  reappeared  in  the  urine  and  fseces,  the  relationship  was 
8.1:1.  In  starvation,  as  shown  by  the  compilation  of  R.  Tigerstedt,^ 
the  phosphorized  constituents  of  the  body  are  destroyed  to  a  much  greater 
extent  than  when  food  is  given  ver}'  poor  in  phosphorus.  In  starvation 
this  relationsliip  is  changed,  namely,  relatively  more  phosphoric  acid  is 
eliminated,  which  seems  to  indicate  that  besides  flesh  and  related  tissues 
another  tissue  rich  in  phosphorus  is  largely  destroyed.  The  starvation 
experiments  show  that  this  is  the  bone-tissue.  According  to  Preysz, 
Olsavszky,  Klug,  and  I.  ]Munk4  the  elimination  of  phosphoric  acid  is 
considerably  increased  by  intense  muscular  work. 

As  the  phosphoric  acid  is  in  part  derived  from  the  nucleins  it  would  be 
expected  that  in  those  diseases  in  which  the  excretion  of  alloxuric  bodies 
was  increased  the  phosphoric  acid  w^ould  also  be  augmented.  This  is  not 
the  case,  and  indeed  we  have  observed  cases  with  an  increased  elimination 
of  alloxuric  l^odies  with  a  diminution  in  the  phosphoric-acid  excretion. 
Cases  of  leucaemia  have  been  observed  in  which  the  phosphoric-acid  excre- 
tion was  reduced,  although  there  was  a  pronounced  increase  in  the  number 
of  leucocytes.  In  these  cases  there  may  be  a  subsequent  excretion  or 
retention  of  phosphoric  acid.  This  last  condition  occurs  also  in  inflamma- 
tory and  renal  diseases.  The  earthy  phosphates  of  the  urine  sometimes 
have  the  tendency  of  precipitating  either  spontaneously  or  after  warming, 
and  this  has  been  called  phosphaturia.  We  are  dealing  here  with  a  dimin- 
ished acidity  and,  it  seems,  Avith  a  diminished  excretion  of  phosphoric  acid 

'  Ehrstrom,  Skand.  Arch.  f.  Physiol.,  14;  Bergmann,  Arch.  f.  exp.  Path.  u.  Pharm., 
47. 

^  Physiologie  des  allgemeinen  Stofifwechsels  und  der  Ernahrung  in  L.  Hermann's 
Handbuch,  fl,  Thl.  1,  79. 

'  Skand.  Arch.  f.  Physiol.,  16. 

*  Preysz,  see  Maly's  Jahresbcr.,  21;  Olsavszky  and  Kkig,  Pfliiger's  Arch.,  54; 
Munk   Arch.  f.  (Anat.  u.)  Physiol.,  1895. 


ESTIMATION    OF    PHOSPHATES.  621 

and  an  increased  elimination  of  lime,  or  at  least  an  essentially  different 
relationship  between  the  phosphoric  acid  and  the  alkaline  earths  of  the  urine, 
as  compared  with  the  normal  (Paxek,  Iwanoff,  Soetber  and  Krieger).^ 

Quantitative  Estimation  of  the  Total  Phosphoric  Acid  in  the  Urine.  This 
estimation  is  most  simply  performed  by  titrating  with  a  solution  of  ura- 
nium acetate.  The  principle  of  the  titration  is  as  follows:  A  warm  solu- 
tion of  phosphates  containing  free  acetic  acid  gives  a  whitish-yellow  pre- 
cipitate of  uranium  phosphate  with  a  solution  of  a  uranium  salt.  This 
precipitate  is  insoluljle  in  acetic  acid,  but  dissolves  in  mineral  acids,  and 
on  this  account  there  is  always  added,  in  titrating,  a  certain  quantit}^  of 
sodium-acetate  solution.  Potassium  ferrocyanide  is  used  as  the  indicator, 
which  does  not  act  on  the  uranium-phosphate  precipitate,  but  gives  a 
reddish-brown  precipitate  or  coloration  in  the  presence  of  the  smallest 
amount  of  soluble  uranium  salt.  The  solutions  necessary  for  the  titration 
are:  1.  A  solution  of  a  uranium  salt  of  which  each  cubic  centimeter  cor- 
responds to  0.007  gram  P2O5  and  which  conains  20.3  grams  of  uranium 
oxide  per  liter.     20  c.c.  of  this  solution  corresponds  to  0.100  gram  P2O5. 

2.  A  solution  of  sodium  acetate.  3.  A  freshly  prepared  solution  of  potas- 
sium ferrocyanide. 

The  uranium  solution  is  prepared  from  uraniimi  nitrate  or  acetate.  Dissolve 
about  35  grams  uranium  acetate  in  water,  add  some  acetic  acid  to  facilitate  solu- 
tion, and  dilute  to  1  liter.  The  strength  of  this  solution  is  determined  b}^  titrating 
with  a  solution  of  sodium  phosphate  of  known  strength  (10.085  grams  crystallized 
salt  in  1  liter,  which  corresponds  to  0.100  gram  PoO^  in  50  c.c).  Proceed  in  the 
same  way  as  in  the  titration  of  the  urine  (see  below),  and  correct  the  solution  by 
diluting  with  water,  and  titrate  again  until  20  c.c.  of  the  uranium  solution  corre- 
sponds exactly  to  50  c.c.  of  the  above  phosphate  solution. 

The  sodium-acetate  solution  should  contain  10  grams  sodium  acetate  and 
10  grams  cone,  acetic  acid  in  100  c.c.  For  each  titration  5  c.c.  of  this  solution 
is  used  with  50  c.c.  of  the  urine. 

In  performing  the  titration,  mix  50  c.c.  of  filtered  urine  in  a  beaker 
with  5  c.c.  of  the  sodium  acetate,  cover  the  beaker  with  a  watch-glass,  and 
warm  over  the  water-bath.  Then  allow  the  uranium  solution  to  flow  in 
from  a  burette,  and  when  the  precipitate  does  not  seem  to  increase,  place 
a  drop  of  the  mixture  on  a  porcelain  plate  with  a  drop  of  the  potassium- 
ferrocyanide  solution.  If  the  amount  of  uranium  solution  added  has  not 
been  sufhcient,  the  color  will  remain  pale  j-ellow  and  more  uranium  solution 
must  be  added ;  but  as  soon  as  the  slightest  excess  of  uranium  solution  has 
been  used  the  color  becomes  a  faint  reddish  brown.  When  this  point  has 
been  obtained,  warm  the  solution  again  and  add  another  drop.  If  the  color 
remains  of  the  same  intensity,  the  titration  is  ended :  but  if  the  color  varies, 
add  more  uranium  solution,  drop  by  drop,  until  a  permanent  coloration  is 
obtained  after  warming,  and  now  repeat  the  test  with  another  50  c.c.  of 
the  urine.  The  calculation  is  so  simple  that  it  is  imnecessary  to  give  an 
example. 

'  Panek,  see  Maly's  Jahresber.,  30,  112;  Iwanoff,  Biochem  Centralbl  ,  1,  710; 
Soetber  and  Krieger,  Deutsch.  Arch.  f.  klin.  Med.,  72;  Campani,  Biochem.  Centralbl., 

3,  616;  Tobler,  Arch.  f.  exp.  Path.  u.  Pharm.,  52. 


622  URINE. 

In  the  above  manner  one  determines  the  total  quantity  of  phosphoric 
acid  in  the  urine.  If  we  wish  to  know  the  phosphoric  acid  combined  with 
alkaline  earths  or  with  alkalies,  we  first  determine  the  total  phosphoric 
acid  in  a  portion  of  the  urine  and  then  remove  the  earthy  phosphates  in 
another  portion  by  ammonia.  The  precipitate  is  collected  on  a  filter, 
washed,  transferred  into  a  beaker  with  water,  treated  with  acetic  acid,  and 
dissolved  by  warming.  This  solution  is  now  diluted  to  50  c.c.  with  water, 
and  5  c.c.  sodium-acetate  solution  added,  then  titrated  with  uranium  solu- 
tion. The  difference  between  the  two  determinations  gives  the  quantity 
of  phosphoric  acid  combined  with  the  alkalies.  The  results  obtained  are 
not  quite  accurate,  as  a  partial  transformation  of  the  monophosphates  of 
the  alkaline  earths  and  also  calcium  diphosphate  into  triphosphates  of  the 
alkaline  earths  and  ammonium  phosphate  takes  place  on  precipitating  with 
ammonia,  and  the  method  gives  too  high  results  for  the  phosphoric  acid 
combined  with  alkalies  and  remaining  in  solution. 

Sulphates.  The  sulphuric  acid  of  the  urine  originates  only  to  a  very 
small  extent  from  the  sulphates  of  the  food.  A  disproportionately  greater 
part  is  formed  by  the  burning  within  the  body  of  the  proteins  which  contain 
sulphur,  and  it  is  chiefly  this  formation  of  sulphuric  acid  from  the  proteins 
which  gives  rise  to  the  previously  mentioned  excess  of  acids  over  the  bases 
in  the  urine.  The  quantity  of  sulphuric  acid  eliminated  by  the  urine 
amounts  to  about  2.5  grams  H2SO4  per  day.  As  the. sulphuric  acid  chiefly 
originates  from  the  proteins,  it  follows  that  the  elimination  of  sulphuric 
acid  and  the  elimination  of  nitrogen  nms  nearly  parallel,  and  the  relation- 
ship N:H2S04  is  about  5:1.  A  complete  parallelism  can  hardly  be  ex- 
pected, as  in  the  first  place  a  part  of  the  sulphur  is  always  eliminated  as 
neutral  sulphur,  and  secondly  because  the  small  proportion  of  sulphur  in 
different  protein  bodies  undergoes  greater  variation  as  compared  with  the 
large  proportion  of  nitrogen  contained  therein.  In  general  the  elimination 
of  nitrogen  and  sulphuric  acid  under  normal  and  under  diseased  conditions 
seem  to  nm  rather  parallel.  Sulphuric  acid  occurs  in  the  urine  partly  pre- 
formed (sulphate-sulphuric  acid)  and  partly  as  ethereal,  sulphuric  acid. 
The  first  is  designated  as  ^-  and  the  other  as  5-sulphuric  acid. 

The  quantity  of  total  sulphuric  acid  is  determined  in  the  following  way, 
but  at  the  same  time  the  precautions  described  in  other  works  must  be 
observed.  100  c.c.  of  filtered  urine  is  treated  with  5  c.c.  of  concentrated 
hydrochloric  acid  and  boiled  for  fifteen  minutes.  While  boihng  i)recipitate 
with  2  c.c.  of  a  saturated  BaCU  solution,  and  warm  for  a  little  while  until 
the  l)arium  sulphate  has  completely  settled.  The  precipitate  must  then  be 
washed  with  water  and  also  with  alcohol  and  ether  (to  remove  resinous 
substances),  and  then  treated  according  to  the  usual  method. 

The  separate  determination  of  the  sulphate-sulphuric  acid  and  the 
ethereal-sulphuric  acid  may  be  accomplished,  according  to  Baumann's 
method,  by  first  precipitating  the  sulphate-sulphuric  acid  by  BaCU  from 
the  urine  acidified  with  acetic  acid,  then  decomposing  the  ethereal-sul- 
phuric acid  by  boiUng  after  the  addition  of  hydrochloric  acid,  and  finally 


POTASSIUM,  SODIUM    AND   AMMONIA.  623 

determining  the  sulphuric  acid  set  free  as  barium  sulphate.  A  still  better 
method  is  the  following,  suggested  by  Salkowski;! 

2U0  c.c.  of  urine  is  precipitated  by  an  ec^ual  volume  of  a  barium  solution 
which  consists  of  2  vols,  barium  hydrate  and  1  vol.  barium-chloride  solu- 
tion, both  saturated  at  the  ordinary  temperature.  Filter  through  a  dry 
filter,  measure  off  100  c.c.  of  the  filtrate  which  contains  only  the  ethereal- 
sulphuric  acid,  treat  with  10  c.c.  of  hydrochloric  acid  of  a  specific  gravity 
1.12,  boil  for  fifteen  minutes,  and  then  warm  on  the  water-bath  until  the 
precipitate  has  completely  settled  and  the  supernatant  liquid  is  entirely 
clear.  Filter  and  wash  with  warm  water  and  with  alcohol  and  ether,  and 
proceed  according  to  the  generally  prescribed  method.  The  difference 
between  the  ethereal-sulphuric  acid  found  and  the  total  quantity  of  sulphuric 
acid  as  determined  in  a  special  portion  of  urine  is  taken  to  be  the  quantity 
of  sulphate-sul])huric  acid. 

FoLiN  2  has  suggested  a  method  for  estimating  the  sulphate-sulphuric 
acid  as  well  as  the  ethereal-sulphuric  acid,  and  also  the  total  sulphur,  which 
is  somewhat  different  from  the  ordinary  methods. 

Nitrates  occur  in  small  quantities  in  human  urine  (Schonbein),  and  they 
probably  originate  from  the  drinking-water  and  the  food.  According  to  Weyl 
and  Citron,^  the  quantity  of  nitrates  is  smallest  with  a  meat  diet  and  greatest 
with  vegetable  food.     The  average  amount  is  about  42.5  milligrams  per  liter. 

Potassium  and  Sodium.  The  quantity  of  these  bodies  eliminated  by  the 
urine  by  a  healthy  adult  on  a  mixed  diet  is,  according  to  Salkowski,^  3-4 
grams  K2O  and  5-6  grams  Na20,  with  an  average  of  about  2-3  grams 
K2O  and  4-6  grams  Na20.  The  proportion  of  K  to  Na  is  ordinarily  3:5. 
The  quantity  depends  above  all  upon  the  food.  In  starvation  the  urine 
may  become  richer  in  potassium  than  in  sodium,  which  results  from  the 
lack  of  common  salt  and  the  destruction  of  tissue  rich  in  potassium.  The 
quantity  of  potassium  may  be  relatively  increased  during  fever,  while  after 
the  crisis  the  reverse  is  the  case. 

The  quantitative  estimation  of  these  bodies  is  performed  by  the  gravi- 
metric methods  as  described  in  works  on  quantitative  analysis.  In  the 
determination  of  the  total  alkalies  recently  new  methods  have  been  de- 
vised by  Pribram  and  Gregor,  and  for  the  potassium  alone  a  method  by 
AuTENRiETH  and  Bernheim.5 

Ammonia.  Some  ammonia  is  habitually  found  in  human  urine  and  in 
that  of  carnivora.  As  above  stated  (page  551),  on  the  formation  of  urea 
from  ammonia,  this  quantity  may  represent  the  small  amount  of  ammonia 

*  Baumann,  Zeitschr.  f.  physiol.  Chem.,  1;    Salkowski,  Virchow's  Arch.,  79. 

^  Journ   of  Biol.  Chem  ,  1,  and  Amer.  Journ.  of  Physiol.,  13. 

'  Schonbein,  Journ.  f.  prakt.  Chem.,  92;  Weyl,  Virchow's  Arch.,  96,  with  Citron, 
ibid.,  101. 

'  Ibid.,  53. 

'  Pribram  and  Gregor,  Zeitschr.  f .  analyt.  Chem.,  38;  Autenrieth  and  Bernheim, 
Zeitschr  f.  physiol.  Chem.,  37. 


€24  URINE. 

which,  is  excluded  from  the  synthesis  to  urea  by  being  combined  with 
acids  formed  in  excess  by  combustion  and  not  united  with  tlie  fixed  alka- 
lies. This  view  is  confirmed  by  the  observations  of  Coranda,  who  found 
that  the  elimination  of  ammonia  was  smaller  on  a  vegetable  diet  and 
larger  on  a  rich  meat  diet  than  on  a  mixed  diet.  On  a  mixed  diet  the 
average  amount  of  ammonia  eliminated  liy  the  urine  is  about  0.7  gram 
NH3  per  day  (Neubauer),  corresponding  to  4.6-5.6  per  cent  of  the  total 
nitrogen  of  the  urine  according  to  Camerer,  Jr.  As  above  stated,  all  the 
ammonia  of  the  urine  is  not  represented  by  the  residue  which  has  eluded 
synthesis  iinto  urea  by  neutralization  with  acids,  becaues,  as  shown  by 
Stadelmann  and  Beckmann,^  ammonia  Ls  ehminated  by  the  urine  even 
during  the  continuous  administration  of  fixed  alkalies. 

Ammonia  exists  on  an  average  of  about  0.90  milligram  in  100  c.c.  of 
human  blood,  and  in  different  amounts  in  all  the  tissues  thus  far  investi- 
gated.2  According  to  Nencki  and  Zaleski  ^  it  is  abundantly  formed  in 
the  cells  of  the  digestive  glands,  the  stomach,  the  pancreas,  and  the  intes- 
tinal mucosa  (of  dogs)  at  the  time  when  protein  foods  are  being  digested 
and  transported  to  the  liver.  As  the  ammonia  introduced  into  the  liver  is 
transformed  into  urea  (see  above),  we  can  therefore  expect  that  in  certain 
diseases  of  the  liver  an  increased  elimination  of  ammonia  and  a  decreased 
excretion  of  urea  will  occur.  In  how  far  this  is  true  has  already  been 
stated  (page  554),  and  we  refer  to  the  researches  of  the  various  authors 
there  cited. 

In  man  and  certain  animals  the  elimination  of  ammonia  is  increased 
by  the  introduction  of  mineral  acids ;  and,  as  shown  by  Jolin,*  organic  acids, 
such  as  benzoic  acid,  which  are  not  destroyed  in  the  body  act  in  a  similar 
manner.  The  ammonia  set  free  in  the  protein  destruction  is  in  part  used 
in  the  neutralization  of  the  acids  introduced,  and  in  this  way  a  destructive 
removal  of  fixed  alkalies  is  prevented.  This  unequal  behavior  of  different 
animals  towards  acidosis  has  been  discussed  in  the  previous  pages. 

Acids  formed  in  the  destruction  of  proteins  in  the  body  act  on  the  elim- 
ination of  ammonia  like  those  introduced  from  without.  For  this  reason 
the  quantity  of  ammonia  in  human  urine  is  increased  under  such  conditions 
and  in  such  diseases  where  an  increased  formation  of  acid  takes  place 

'  Coranda,  Arch,  f  exp.  Path.  u.  Pharm.,  12;  Stadelmann  (and  Beckmann),  Ein- 
fluss  der  Alkalien  auf  den  Stoffwechsel,  etc.  Stuttgart,  1890;  Camerer,  Zeitschr.  f. 
Biologic,  43. 

^  See  Salaskin,  Zeitschr  f  physio!  Chem.,  25,  449,  and  foot-notes  1  and  2,  page 
241. 

'  Arch  des  science  biol.  de  St  Petersbourg,  4,  and  Salaskin,  J.  c.  See  also  Nencki 
and  Zaleski,  Arch.  f.  exp   Path,  u   Pharm.,  37. 

*  John,  Skand.  Arch.  f.  Physiol.,  1.  In  regard  to  the  behavior  of  ammonium  salts 
in  the  animal  body,  see  Rumpf  and  Kleine,  Zeitschr.  f.  Biologic,  34,  and  the  works 
cited  on  page  5.'j4. 


CALCIUM  AND  MAGNESIUM.  625 

because  of  an  increased  metabolism  of  proteins.  This  is  the  case  with  a 
lack  of  oxygen  in  fevers  and  diabetes.  In  the  last-mentioned  disease 
organic  acids,  ^-oxybutyric  acid  and  acetoacetic  acid,  are  produced,  wliich 
pass  into  the  urine  combined  with  ammonia.^  Other  observations  also 
indicate  that  the  .elimination  of  ammonia  by  the  urine  is  increased  on  insuf- 
ficient or  diminished  supply  of  alkahes  or  alkaline  earths. 

The  detection  and  quantitative  estimation  of  ammonia  used  to  be  performed 
according  to  the  method  suggested  by  Schlosixg.  The  principle  of  this  method 
is  that  the  ammonia  from  a  measured  amount  of  urine  is  set  free  by  lime-water 
in  a  closed  vessel  and  absorbed  by  a  measured  amount  of  X/10  sulphuric  acid. 
After  the  absorption  of  the  ammonia  the  quantity  is  determined  by  titrating  the 
remaining  free  sulphmic  acid  with  a  X/10  caustic-alkali  solution.  This  method 
gives  low  results,  and  in  exact  work  we  must  proceed  as  suggested  by  Bohland.* 

The  recent  methods  for  estimating  the  ammonia  are  all  based  upon  the 
distillation  of  the  ammonia,  after  the  addition  of  lime,  magnesia,  or  alkaU 
carbonate,  at  low  temperatures  either  by  the  aid  of  vacuum  (Nenxki  and 
Zaleski,  Wurster,  Kruc;er,  Reich,  and  Schittexhelm,  and  Schaffer) 
or  by  the  aid  of  a  current  of  air  (Folin)  and  then  collecting  it  in  a  standard 
acid. 

According  to  the  methods  suggested  by  KRiJGER,  Reich  and  Schitten- 
helm^  25  c.c.  of  the  urine  is  placed  in  a  distillation-flask  with  about  10 
grams  of  NaCl  and  1  gram  of  Na2C03,  and  this  distilled  at  43°  C.  and  a 
pressure  of  30-40  millimeters  Hg  with  the  aid  of  an  air-pump.  Alcohol 
is  added  to  prevent  foaming.  The  ammonia  is  absorlTed  in  X/10  acid 
contained  in  a  Peligot  tube  siuTounded  by  ice-water,  and  when  the 
distillation  is  finished  the  acid  is  retitrated.  making  use  of  rosohc  acid  as 
indicator.  In  regard  to  details,  see  the  original  pubhcations.  Schaffer 's 
method  is  practically  the  same. 

Calcium  and  magnesium  occur  in  the  urine  chiefly  as  phosphates. 
The  quantity  of  earthy  phosphates  eUminated  daily  is  somewhat  more 
than  1  gram,  and  of  this  amount  |  is  magnesium  and  J  calcium  phos- 
phate. This  statement,  as  found  by  Renw^^ll  and  Gross,^  is  not  correct. 
or  at  least  is  not  valid  in  general,  as  they  found  more  calcium  than  mag- 
nesium in  the  urine.  In  acid  urines  the  mono-  as  well  as  the  dihydrogen 
earthy  phosphates  are  found,  and  the  solubihty  of  the  first,  among  which 
the  calcium  salt  CaHP04  is  especially  insoluble,  is  particularly  augmented 
by  the  presence  in  the  urine  of  dihydrogen  alkali  phosphates  and  sodium 
chloride   COtt^).     The  quantity  of  alkahne  earths  in  the  urine  depends 

'  On  the  elimination  of  ammonia  in  disease,  see  the  recent  works  of  Rumpf,  Vir- 
chow's  Arch.,  134;    Hallervorden,  ibid. 

'Pfliiger's  Arch.,  43,  32 

'  Zeitschr.  f  physiol.  Chem.,  89;  Schaffer,  Amer.  Journ.  of  Physiol.,  8,  which  con- 
tains the  Uterature. 

*  Renwall,  Skand.  Arch.  f.  Physiol  ,  16;    Gross,  Biochem.  Centralbl.,  4,  ISO. 

^Zeitschr.  f.  pliysiol.  Chem.,  10 


626  URINE. 

on  the  composition  of  the  food.  The  Ume-salts  absorbed  are  in  great  part 
excreted  again  into  the  intestine,  and  the  quantity  of  lime-salts  in  the  urine 
is  therefore  no  measure  of  the  absorption  of  the  same.  The  introduction 
of  readily  soluble  lime-salts  or  the  addition  of  hydrochloric  acid  to  the  food 
may  therefore  cause  an  increase  in  the  quantity  of  lime  in  the  urine,  while 
the  reverse  takes  place  on  adding  alkali  phosphate  to  the  food.  Nothing 
is  known  with  positiveness  in  regard  to  the  constant  and  regular  change 
in  the  elimination  of  calcium  and  magnesium  salts  in  disease/  and  in  these 
conditions  the  excretion  is  chiefly  dependent  upon  the  diet,  and  upon  the 
formation  and  introduction  of  acid. 

The  quantity  of  calcium  and  magnesium  is  determined  according  to  the 
ordinary  well-known  methods. 

Iron  occurs  in  the  urine  only  in  small  quantities,  and,  as  it  seems  from  the 
investigations  of  Kunkel,  Giacosa,  Robert  and  his  pupils,  it  does  not  exist 
as  a  salt,  but  as  an  organic  combination — in  part  as  pigment  or  chromogen.  The 
statements  in  regard  to  the  iron  present  seem  to  show  that  the  quantity  is  very 
variable,  from  1  to  11  milligrams  per  liter  of  urine  (^Iagnier,  Gottlieb,  Robert 
and  his  pupils).  Jolles  found  as  an  average  for  twelve  persons  8  milligrams  of 
iron  in  twenty-four  hours,  while  Hoffmann,  Neumann  and  Mayer  ^  found  lower 
results — an  average  of  1.09  and  0.983  milligrams.  The  quantity  of  silicic  acid  is 
ordinarily  stated  to  amount  to  about  0.3  p.  m.  Traces  of  hydrogen  peroxide  also 
occur  in  the  urine. 

The  gases  of  the  urine  are  carbon  dioxide,  nitrogen,  and  traces  of 
oxygen.  The  quantity  of  nitrogen  is  not  quite  1  vol.  per  cent.  The  carbon 
dioxide  varies  considerably.  In  acid  urines  it  is  hardly  one  half  as  great 
as  in  neutral  or  alkaline  urines. 


IV.    The  Quantity  and  Quantitative  Composition  of  Urine. 

The  quantity  and  composition  of  urine  is  liable  to  great  variation. 
The  circumstances  which  under  physiological  conditions  exercise  a  great 
influence  are  the  follo^^'ing:  the  blood-pressure,  and  the  rapidity  of  the 
blood-current  in  the  glomeruli;  the  quantity  of  urinar}^  constituents, 
especially  water  in  the  blood;  and,  lastly,  the  condition  of  the  secretory 
glandular  elements.  Above  all,  the  quantity  and  concentration  of  the 
urine  depend  on  the  quantity  of  water  which  is  introduced  into  the  blood  or 
which  leaves  the  body  in  other  ways.  The  excretion  of  urine  is  increased  by 
drinking  freely  or  by  reducing  the  quantity  of  water  othen\ase  removed; 
and  it  is  decreased  by  a  diminished  ingestion  of  water  or  by  a  greater  loss 

1  See  Albu  and  Neuberg,  1.  c. 

^Kunkel,  cited  from  Maly's  Jahresber.,  11;  Giacosa,  ibid.,  16;  Robert,  Arbeiten 
des  Pharm.  Inst,  zu  Dorpat,  7;  Magnier,  Ber,  d.  deutsch.  chem.  Gesellsch.,  7;  Gott- 
lieb, Arch.  f.  exp.  Path.  u.  Pharm.,  26;  Jolles,  Zeitschr.  f.  anal.  Chem  ,  36;  Hoffmann, 
Zeitschr.  f.  anal  Chem.,  40;   Neumann  and  Mayer,  Zeitschr.  f.  physiol.  Chem.,  37. 


QUANTITY  AND  QUANTITATIVE  COMPOSITION  OF  URINE.    627 

of  water  in  other  ways.  Ordinarily  in  man  just  as  much  water  is  eliminated 
by  the  kidneys  as  by  the  skin,  lungs,  and  intestine  together.  At  lower 
temperatures  and  in  moist  air,  since  under  these  conditions  the  elimination 
of  water  by  the  skin  is  diminished,  the  excretion  of  urine  may  be  con- 
siderably increased.  Diminished  introduction  of  water  or  increased  elimi- 
nation of  water  by  other  ways— as  in  ^iolent  diarrhoea  or  vomiting,  or  in 
profuse  perspiration — greatl}^  diminishes  the  amount  of  urine  excreted. 
For  example,  the  urine  may  sink  as  low  as  500-400  c.c.  per  day  in  intense 
summer  heat,  while  after  copious  draughts  of  water  the  elimination  of 
3000  c.c.  of  urine  has  been  ol)served  during  the  same  time.  The  quantity 
of  urine  voided  in  the  course  of  twenty-four  hours  varies  considerably 
from  day  to  day,  the  average  being  ordinarily  calculated  as  1500  c.c.  for 
healthy  adult  men  and  1200  c.c.  for  women.  The  minimum  elimination 
occurs  during  the  early  morning,  between  2  and  4  o'clock;  the  maximum, 
in  the  first  hours  after  waking  and  from  1-2  hours  after  a  meal. 

The  quantit}'  of  solids  excreted  per  day  is  nearly  constant,  even  though 
the  quantity  of  urine  may  vary,  and  it  is  quite  constant  when  the  manner  of 
li^'ing  is  regular.  Therefore  the  percentage  of  soUds  in  the  urine  is  natu- 
rally in  inverse  proportion  to  the  quantit}-  of  urine.  The  average  amount 
of  solids  pert  wenty-four  hours  is  calculated  as  60  grams.  The  quantity 
may  be  calculated  with  approximate  accuracy  from  the  specific  gra^'ity 
if  the  second  and  third  decimals  of  this  factor  be  multiplied  b}'  Haser's 
coefficient,  2.33.  The  product  gives  the  amount  of  solids  in  1000  c.c. 
of  urine,  and  if  the  quantity  of  urine  eUminated  in  twenty -four  hours  be 
measured,  the  quantity  of  solids  in  twent3--four  hours  may  be  easil}-  calcu- 
lated. For  example,  1050  c.c.  of  urine  of  a  specific  gravity  1.021  was  elimi- 
nated in  twenty-four  hours;  therefore  the  quantity  of  solids  excreted  was 

48  9x  1050 
21x2.33  =  48.9  and       "         — =51.35   grams.     Long  i  has   made   a  new 

determination  of  the  coefficient  for  a  specific  gra\'ity  taken  at  25°  C.  and 
finds  that  it  is  equal  to  2.6,  which  corresponds  nearly  to  Haser's  coefficient 
at  15°  C. 

Those  bodies  which,  under  physiological  conditions,  affect  the  density 
of  the  urine  are  common  salt  and  urea.  The  specific  gra^'ity  of  the  first  is 
2.15  and  the  last  only  1.32,  so  it  is  easy  to  undei*stand,  when  the  relative 
proportion  of  these  two  bodies  essentially  de\'iates  from  the  normal,  why 
the  above  calculation  from  the  specific  gra^'ity  is  not  exact.  The  same  is 
true  when  a  urine  poor  in  normal  constituents  contains  large  amounts  of 
foreign  bodies,  such  as  albumin  or  sugar. 

As  above  stated,  the  percentage  of  solids  in  the  urine  generally  decreases 
wdth  a  greater  elimination,   and  a  \ery  considerable  excretion  of  urine 

'  Joum.  Amer.  Chem.  Soc,  25. 


628  URINE. 

{polyuria)  has  therefore,  as  a  rule,  a  lower  specific  gravity.  An  important 
exception  to  this  rule  is  observed  in  urine  containing  sugar  (diabetes  melli- 
His),  in  which  there  is  a  copious  excretion  with  a  ven-  high  specific  gravity- 
due  to  the  sugar.  In  cases  where  very  little  urine  is  excreted  {oliguria), 
e.g.,  during  profuse  perspiration,  in  diarrhoea,  and  in  fevers,  the  specific 
gra^4ty  of  the  urine  is  as  a  rule  xery  high;  the  percentage  of  solids  is  also 
high  and  the  urine  lias  a  dark  color.  Sometimes,  as  for  example  in  certain 
cases  of  albuminuria,  the  urine  may  have  a  low  specific  gravity  notwith- 
standing the  oUgTiria,  and  be  poor  in  solids  and  light  in  color. 

In  certain  cases  it  is  interesting  to  know  the  relationship  between  the 
carbon  and  the  nitrogen,  or  the  quotient  C/N.  This  factor  may  vary 
between  0.7  and  1;  as  a  mle,  it  amounts  on  an  average  to  0.87,  but  changes 
according  to  the  nature  of  the  food  and  is  higher  alter  a  diet  rich  in  carbo- 
hydrates than  after  food  rich  in  fat  (Pregl,  Tangl,  Langstein  and  Steinitz, 
and  others  ^). 

It  is  difficult  to  give  a  tabular  view  of  the  composition  of  urine  on 
account  of  its  variation.  For  certain  purposes  the  following  table  may  be 
of  some  value,  but  it  must  not  be  overlooked  that  the  results  are  not  given 
for  1000  parts  of  urine,  but  only  approximate  figiu'es  for  the  quantities  of 
the  most  important  constituents  which  are  eliminated  during  the  course  of 
twenty-four  hours  in  a  volume  of  1500  c.c.  of  urine.  These  figures  apply 
only  to  a  diet  which  corresponds  to  Voit's  standard  figures,  namely  118 
grams  protein,  56  grams  fat,  and  500  grams  carbohydrate  per  day,  and  to 
a  man  of  average  weight. 

Daily  quantity  of  solids  =  60  grams. 
Organic  constituents  =  35  grams.  Inorganic  constituents  =  25  grams. 

Urea 30.0  grams.         Sodium  chloride  (NaCl)  .  . .    15.0  grams. 

Uric  acid 0.7       "  Sulphuric  acid  (H^SO,)  ...      2.5  " 

Creatinine 1.5       "  Phosphoric  acid  (P.,05).  .  .  .      2.5  " 

Hippuric  acid 0.7       "  Potash  (ICO) * 3.3  " 

Remaining  organic  bodies. .     2.1       "  Ammonia  (NH3) 0.7  " 

Magnesia  (MgO)  1  f.  ^  ,, 

Lime  (CaO)  / "•* 

Remaining  inorganic  bodies    0.2  " 

Urine  contains  on  an  average  40  p.  m.  solids.  The  quantity  of  urea  is 
about  20  p.  m.,  and  common  salt  about  10  p.  m. 

The  physico-chemical  methods  are  being  used  in  urinary  analysis  even 
to  a  greater  extent  than  in  the  analysis  of  other  animal  fluids.  A  great 
number  of  cry^oscopic  determinations  but  fewer  conductivity  determinations 
have  been  made.  A  constant  relationship  between  the  values  found  by 
phj'sico-chemical  methods  and  the  analytical  methods  has  been  sought, 
for  example,  between  the  freezing-point  depression  and  the  specific  gravity 

*  Pregl,  Pfliiger's  Arch  ,  75,  which  contains  the  older  literature.  Tangl,  Arch.  f. 
(Anat.  u.)  Physiol.,  1899,  Suppl.;   Langstein  and  Steinitz,  Centralbl.  f.  Physiol.,  19. 


CASUAL  URINARY   CONSTITUENTS.  629 

or  the  common  salt  and  others ;  or  attempts  have  been  made  to  find  certain 
constants  in  the  composition  of  the  urine  based  upon  the  results  of  various 
methods,  and  in  this  way  to  obtain  an  explanation  as  to  the  mechanism  of 
the  excretion  of  urine  in  order  to  apply  them  for  diagnostic  purposes.  The 
results  obtained  are.  as  is  to  be  expected,  so  variable  and  dependent  upon 
so  many  conditions  which  cannot  be  controlled  that  definite  conclusions 
must  be  drawn  with  the  greatest  caution.  In  regard  to  the  value  and  use- 
fulness of  the  various  constants  and  relations  which  are  based  upon  theo- 
retical considerations,  the  views  are  unfortunateh'  still  too  divergent. 

V.   Casual  Urinary  Constituents. 

The  casual  appearance  in  the  urine  of  medicinal  agents  or  of  urinary  con- 
stituents resulting  from  the  introduction  of  foreign  substances  into  the 
organism  is  of  practical  importance,  because  such  compounds  may  inter- 
fere in  certain  urinar>'  investigations;  they  also  afford  a  good  means  of 
determining  whether  certain  substances  have  been  introduced  into  the 
organism  or  not.  From  this  point  of  \aew  a  few  of  these  bodies  will  be 
spoken  of  in  a  following  section  (on  the  pathological  urinarv^  constituents). 
The  presence  of  these  foreign  bodies  in  the  urine  is  of  special  interest  in 
those  cases  in  which  they  serve  to  elucidate  tlie  chemical  transformations 
which  certain  substances  undergo  within  the  organism.  As  inorganic 
substances  generally'  leave  the  body  unchanged, ^  they  are  of  very  Httle 
interest  from  this  standpoint;  but  the  changes  which  certain  organic  sub- 
stances undergo  when  introduced  into  the  animal  body  may  be  studied  b}' 
the  transformation  products  as  found  in  the  urine. 

The  bodies  belonging  to  the  fatty  series  undergo,  though  not  without 
exceptions,  a  combustion  leading  towards  the  final  products  of  metab- 
olism; still,  often  a  greater  or  smaller  part  of  the  bodies  in  question  escape 
oxidation  and  appear  unchanged  in  the  urine.  A  part  of  the  acids  belong- 
ing to  this  series,  which  are  otherwise  decomposed  into  water  and  carbonates 
and  render  the  urine  neutral  or  alkaline,  may  act  in  this  manner.  The 
volatile  fatty  acids  poor  in  carbon  are  less  easily  oxidized  than  those  rich  in 
carbon,  and  they  therefore  pass  unchanged  into  the  urine  in  large  amounts. 
This  is  especially  true  of  formic  and  acetic  acids  (Schotten,  Grehant  and 
QuiNQUAUD^).  The  statements  in  regard  to  oxahc  acid  are  contradictory. 
In  birds,  according  to  Gaglio  and  Giunti,  it  is  not  oxidized.  In  mammals 
it  is  in  great  part  oxidized,  according  to  Giuxti,  while  Gaglio  and  Pohl 


'  In  regard  to  the  behavior  of  certain  of  these  bodies,  see  Heffter,  Die  Ausscheidung 
korperfremden  Substanzen  im  Harn,  Er^ebnisse  d.  Physiol.,  2,  Abt.  1. 

^  Scliotten,  Zeitschr.  f.  physiol.  Chem.,  7;  Grehant  and  Quinquaud,  Compt.-rend., 
104. 


630  URINE. 

claim  that  it  is  not  destroyed.  Marfori  and  Giuxti  claim  that  in  human 
beings  oxalic  acid  is  in  great  part  oxidized,  although  the  recent  investiga- 
tions of  Salkowski,  Pierallini,  Stradomsky,  Klemperer  and  Trit- 
SCHLER  1  seem  to  show  that  the  acid  is  only  in  part  destroyed  in  the  animal 
body.  In  order  to  exactly  determine  that  portion  of  the  ingested  oxalic 
acid  which  is  absorbed  and  excreted  by  the  urine  or  oxidized  in  the  body,  it 
must  necessarily  be  known  whether  or  not  a  portion  of  the  acid  is  destroyed 
in  the  intestine  and  is  therefore  not  absorbed.  Tartaric  acids  act  differ- 
ently, according  to  Brion;^  thus  in  dogs  the  levotartaric  acid  is  nearly 
entirely  consumed,  while  a  little  more  than  70  per  cent  of  dextrotartaric 
acid  is  burnt.  Racemic  acid  is  oxidized  to  a  still  less  extent  in  the  animal 
body.  Succinic  and  malic  acids  are  completely  combustible,  according  to 
PoHL.3  Examples  of  the  different  behavior  of  stereoisomeric  substances 
have  already  been  given  on  page  109. 

The  acid  amides  appear  not  to  be  altered  in  the  body  (Schultzen  and 
Nencki^).  The  amino-acids  may  indeed,  when  introduced  into  the  body 
in  large  quantities,  be  in  part  eliminated  unchanged  by  the  urine;  but 
otherwise,  as  stated  above  (page  550)  for  leucine,  glycocoll,  and  aspartic 
acid,  they  are  decomposed  w'ithin  the  body,  and  may  therefore  cause  an 
increased  excretion  of  urea.  That  in  the  demolition  of  the  amino-acids 
a  deamidation  takes  place  is  shown  by  alanine  yielding  lactic  acid  and 
diaminopropionic  acid,  yielding  glyceric  acid,  as  mentioned  in  a  previous 
chapter  (VIII).  The  amino-acids  give  an  instructive  example  of  the 
unequal  behavior  of  stereometric  substances  in  the  animal  body,  as  the 
inactive  acids  are  so  changed  and  transformed  that  the  component  foreign 
to  the  body  is  more  or  less  abundantly  excreted,  while  that  occurring  in 
the  body  protein  is  oxidized  (Schittenhelm  and  Katzenstein,  Wohlge- 
muth 5) .  In  connection  with  the  amino-acids  it  is  to  be  recalled  that 
according  to  the  observations  of  Abderhalden  and  Bergell  ^  glycyl- 
glycine  introduced  subcutaneously  in  rabbits  appeared  in  the  urine  as  gly- 
cocoll. 

Various  amino  acids  show  a  somewhat  different  behavior.     Sarcosine 


^Gaglio,  Arch.  f.  exp.  Path.  u.  Pharm.,  22;  Giunti,  Chem.  Centralbl,  1897,  2; 
Marfori,  Maly's  Jahresber.,  20  and  27;  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.,  37;  Sal- 
kowski, Berl.  kHa.  Wochenschr.,  1900;  Pierallini,  Virchow's  Arch.,  160;  Stradomsky, 
ibid.,  163;   Klemperer  and  Tritschler,  Zeitschr.  f.  klin.  Med.,  44. 

^  Zeitsclir.  f.  physiol.  Chem.,  25. 

^  Polil,  Arch.  f.  exp.  Path.  u.  Pharm.,  37,  which  also  contains  the  statements  on 
the  intermediary  products  formed  in  the  oxidation  of  the  fatty  bodies. 

*  Zeitschr.  f .  Biologic,  8. 

*  Schittenhelm  and  Katzenstein,  Zeitschr.  f.  exp.  Path.,  2,  cited  from  Biochem. 
Centralbl.,  5;  Wohlgemuth,  Ber.  d.  d.  chem.  Gesellsch.,  38. 

'  Zeitschr.  f.  physiol.  Clem.,  39. 


CASUAL  URIXARY  CONSTITUENTS.  631 

(methylglycocoll),  (CH3)NH.CH2.COOH,  which  is  not  readily  burnt, 
passes  therefore  in  great  part  unchanged  mto  the  urine ,  but  perhaps  also 
passes  in  small  part  into  the  corresponding  uramino-acid.  methijlhydantoic 
acid,  NH2.CO.N(CH3).CH2.COOH  (Schultzex  i).  Likewise  taurine, 
aminoeth}  Isulphonic  acid,  which  acts  somewhat  differentl}'  in  different 
animals  (Salko wski  2) ,  passes  in  human  beings,  at  least  in  pait,  into  the 
corresponding  uramino-acid,  taurocarhamic  acid.  NH2.CO.NH.C2H4.SO2.OH. 
A  part  of  the  taurine  also  appeai-s  as  such  in  the  urine.  In  rabbits,  when 
taurine  is  introduced  into  the  stomach  nearly  all  its  sulphur  appears  in  the 
urine  as  sulphuric  and  hyposulphurovs  acids.  After  subcutaneous  injection 
the  taurine  appears  again  in  great  part  unchanged  in  the  urine.  In  dogs 
a  great  part  of  the  sulphur  of  cystine  appears  in  the  urine  as  sulphate  (also 
as  thiosulphate)  (Blum,  Abderhaldex  and  Samuely^). 

The  nitriles,  including  hydrocyanic  acid,  pass,  according  to  Laxg,  into 
sulphocyanide  combinations,  and  this  sulphocyanide  apparenth-  originates 
from  the  non-oxidized  sulphur  of  the  proteins,  which  is  readily  split  off. 
Pascheles'  observations  indicate  that,  in  an  alkaline  reaction  and  at  the 
temperature  of  the  body,  this  sulphur  can  convert  the  alkah-cyanides 
readily  into  sulphocyanides.  The  alkali  sulphocj^anides  when  ingested 
are  nearly  quantitatively  eliminated  in  the  urine,  according  to  Pollak.^ 

By  substitution  with  halogens,  bodies  othenvise  readily  oxidizable  are 
converted  into  difficultly  oxidizable  ones.  While  the  aldehydes  are  readily 
and  completely  burnt  like  the  primary'  and  secondarv'  alcohols  of  the  fatty 
series,  the  halogen  substituted  aldehydes  and  alcohols  are,  on  the  contrar}', 
difficultly  oxidizable.  The  halogen  substitution  products  of  methane 
(chloroform,  iodoform,  and  bromoform)  are  at  least  in  part  destroyed  and 
the  corresponding  alkali  compounds  of  the  halogen  pass  into  the  urine.^ 

By  conjugation  mith  sulphuric  acid,  the  alcohols  which  are  othen\'ise 
readily  oxidizable  may  be  guarded  against  combustion,  and  consequently 
the  alkali  salt  of  ethylsulphuric  acid  is  not  burnt  in  the  body  (Salkowski  ^). 

The  organic  combinations  containing  sulphur  act  somewhat  differently. 
W.  Smith  states  that  the  sulphur  of  the  thio-acids,  like  thiogly colic  acid. 


'  Ber.  d.  deutsch.  chem.  Gesellsch.,  5.  See  also  Baumami  and  v.  Mering,  ibid.,  8, 
584,  and  E.  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  4,  107. 

^  Ber.  d.  deutsch.  chem.  Gesellsch.,  6,  and  Virchow's  Arch..  58. 

^  Blum,  Hofmeister's  Beitrage,  o;  Abderhalden  and  Samuely,  Zeitschr.  f.  physiol. 
Chem.,  46. 

^  Lang,  Arch.  f.  exp.  Path.  u.  Pharm.,  34;  Pascheles,  ibid.;  Pollak,  Hofmeister's 
Beitrage,  2. 

^  See  Hamack  and  Griindler,  Berlin,  klin.  Wochenschr.,  1S83;  Zeller,  Zeitschr.  f. 
physiol.  Chem.,  8;  Kast,  ibid.,  11;  Binz,  Arch.  f.  exp.  Path.  u.  Pharm.,  28;  Zeehuisen, 
Maly's  Jahresber.,  23. 

'  Pfliiger's  .-Vrch.,  4. 


632  URINE. 

CH2.SH.COOH,  is  in  part  oxidized  to  sulphuric  acid,  and  according  to 
GoLDMANX  the  same  result  occurs  with  aminothiolactic  acid  (cysteine) 
and  the  sulphur  of  the  thio-alcohols  (ethyl  mercaptans).  On  the 
contrar}^,  ethylsulphide,  sulphonic  and  sulpho  acids  in  general  (Salkowski, 
Smith  i)  are  not  changed  into  sulphuric  acid.  Oxyethylsulphonic  acid, 
HO.C2H4.SO2.OH,  which  is  in  part  oxidized  to  sulphuric  acid,  is  an  ex- 
ception (Salkowski). 

Conjugation  with  glucuronic  acid  occurs,  according  to  the  investigations 
of  SuNDViK  and  especially  of  O.  Neubauer,  in  many  substituted  as  well 
as  non-substituted  alcohols,  aldehydes,  and  ketones.  Chloral  hydrate, 
C2CI3OH -I- H2O,  passes,  after  it  has  been  converted  into  trichlorethyl- 
alcohol  bv  a  reduction,  into  a  levogyrate  reducing  acid,  urochloralic  acid 
or  trichlorethylglucuronic  acid,C2Cl3H2.C6H407  (Musculus  and  V.  Mering). 
Of  the  primar}^  alcohols  investigated  by  Neubauer  2  (upon  rabbits  and 
dogs)  methyl  alcohol  gave  no  conjugated  glucuronic  acid,  and  ethyl  alcohol 
only  a  small  amount.  Isobutyl  alcohol  and  active  amyl  alcohol  yielded 
relativelv  large  quantities.  Secondaiy  alcohols  produced  conjugated  glucu- 
ronic acids,  and  indeed  to  a  greater  extent  than  the  primary  alcohols, 
especially  in  rabbits.  The  ketones  are  reduced  in  part  into  secondary  alco- 
hols and  are  partly  excreted  as  the  conjugated  acid.  This  could  be  shown 
for  acetone  with  rabbits  Imt  not  with  dogs. 

The  homo-  and  heterocyclic  compounds  pass,  as  far  as  is  known,  into 
the  urine  as  such,  or,  after  a  previous  partial  oxidation  or  synthesis  with 
other  bodies,  and  appear  as  so-called  aromatic  compounds.  That  the 
benzene  ring  is  destroyed  in  the  body  in  certain  cases  is  very  prob- 
able. 

The  fact  that  benzene  may  be  oxidized  outside  of  the  body  into  carbon 
dioxide,  oxalic  acid,  and  volatile  fatty  acids  has  been  known  for  a  long 
time;  and  as  in  these  cases  a  rupture  of  the  benzene  ring  must  take  place, 
so  also,  it  must  be  admitted,  when  aromatic  substances  undergo  a  com- 
bustion in  the  animal  body,  a  splitting  of  the  benzene  nucleus  with  the 
formation  of  fatty  bodies  must  be  the  result.  If  this  does  not  occur,  then 
the  benzene  nucleus  is  eliminated  with  the  urine  as  an  aromatic  compound 
of  one  kind  or  another.  As  the  benzene  nucleus  can  protect  a  substance 
belonging  to  the  fatty  series  from  destruction  when  conjugated  with  it. 


'  Smith,  Pfliiger's  Arch.,  53,  55,  57,  and  Zeitschr.  f.  physiol.  Chem.,  17;  Salkowski, 
Virchow's  Arch.,  66;  Pfliiger's  Arch.,  39;  Goldmann,  Zeitschr.  f.  physiol.  Chem.,  9; 
also  Baumann  and  Kast,  ibid.,  1-t. 

^  Sundvik,  Maly's  Jahresber.,  16;  Musculus  and  v.  Mering,  Ber.  d.  deutsch.  chem. 
Gesellsch.,  8;  also  v.  Mering,  ibid.,  15;  Zeitschr.  f.  physiol.  Chem.,  6;  Kiilz,  Pfliiger's 
Arch.,  28  and  33;    0.  Neubauer,  Arch.  f.  exp.  Path.  u.  Pharm.,  46. 


CASUAL  URINARY   CONSTITUENTS.  633 

which  is  the  case  with  the  glycocoU  of  liippuric  acid,  it  seems  that  the 
aromatic  nucleus  itself  may  likewise  be  protected  from  oxidation  in  the 
organism  by  synthesis  with  other  bodies.  The  aromatic  ethereal-sulphuric 
acids  are  examples  of  this  kind. 

The  difficulty  in  deciding  whether  the  benzene  ring  itself  is  destroyed 
in  the  body  lies  in  the  fact  that  we  do  not  know  all  the  different  aromatic 
transformation  products  wliich  may  be  produced  by  the  introduction  of 
any  such  substance  into  the  organism,  and  wliich  must  be  sought  for  in 
the  urine.  On  this  account  it  is  also  impossible  to  learn  by  exact  quanti- 
tative determinations  whether  or  not  an  aromatic  substance  ingested  or 
absorbed  appears  again  unchanged  in  the  urine.  Certain  observations 
render  it  probable  that  the  benzene  ring,  as  above  mentioned,  is  at  least 
in  certain  cases  destroyed  in  the  body.  Schottex,  Baum.\xn,  and  others 
have  found  that  certain  amino-acids,  such  as  phenylamino-propionic 
add,  amino-cinnamic  acid,  and  tyrosine,  when  introduced  into  the  body 
cause  no  increase  in  the  quantity  of  known  aromatic  substances  in  the 
urine;  this  makes  a  destruction  of  these  amino-acids  in  the  animal  body 
seem  probable.  According  to  F.  Kxoop  ^  phenyl-a-lactic  acid  and  phenyl- 
oi-ketopropionic  acid  (phenyl  pyroracemic  acid)  have  a  similar  beha\ior, 
while  Juv.^lta's  statement  that  phthaHc  acid  is  destroyed  in  the  animal 
body  is  denied  by  E.  Pribra:m.2  The  benzene  derivatives  vary  in  behavior 
according  to  the  position  of  the  substitution,  for,  as  found  by  R.  Cohx,^ 
among  the  di-derivatives  the  ortho-compounds  are  more  readily  destroyed 
than  the  corresponding  meta-  or  para-compounds. 

An  oxidation  in  the  side  chain  of  aromatic  compounds  is  often  found 
and  may  also  occur  in  the  nucleus  itself.  As  an  example,  benzene  is  first 
oxidized  to  oxybenzene  (Schultzex  and  Naunyn),  and  this  is  then  further 
in  part  oxidized  into  dioxyhenzenes  (Baumaxx  and  Preusse).  Naphtha- 
lene appears  to  be  converted  into  oxy naphthalene,  and  probably  a  part 
also  into  dioxynaphthalene  (Lesnik  and  ^I.  Nexcki).  The  hydrocarbon 
with  an  amino-  or  imino-group  may  also  be  oxidized  by  a  substitution  of 
hydroxyl  for  hydrogen,  especially  when  the  formation  of  a  derivative  in 
the  para-position  is  possible  (Klixgenberg).  For  example,  aniline, 
C6H5.XH2,  passes  into  paraminophenol,  which  latter  passes  into  the 
urine  as  its  ethereal-sulphuric  acid,  H2N.C6H4.O.SO2.OH  (F.  ^Iuller). 
Acetanilid  is  in  part   converted  into  acetyl  paraminophenol    (Jaff:^  and 


'  Schotten,  Zeitschr.  f.  physiol.  Chem.,  7  and  8;  Baumann,  ibid.,  10,  130.  In  regard 
to  the  behavior  of  tyrosine,  see  especially  Blendermann,  ibid.,  6;  Schotten,  ibid.  7; 
Bass,  ibid.,  11;  and  R.  Cohn,  ibid.,  11;  F.  Iviioop,  Der  Abbau  aromatischer  Fett- 
sauren  im  Tierkorper,  Habilit.-Schrift,  Freiburg,  1904. 

'  Juvalta,  Zeitschr.  f  physiol.  Chem.,  13;  Pribram,  Arch.  f.  exp.  Path.  u.  Pharm.  51. 

•  Zeitschr.  f.  physiol.  Chem.,  1". 


634  URINE. 

HiLBERT,    K.    Morner),     and     carhazol    into    oxycarbazol     (Klingen- 

BERG  ^). 

An  oxidation  of  the  side  chdin  may  occur  by  the  hydrogen  atoms  being 
replaced  by  hydroxy!,  as  in  the  oxidation  of  indol  and  skatol  into  indoxyl 
and  skatoxyl.  An  oxidation  of  tlie  side  chain  may  also  take  place  with  the 
formation  of  carboxyl;  thus,  for  example.  foZwene,  C6H5.CH3  (Schultzen 
and  Naunyn),  ethyl-henzene,  C6H5.C2H5,  and  ipropylhenzene,  C6H5.C3H7 
(Nencki  and  Giacosa^),  besides  many  other  bodies,  are  oxidized  into 
benzoic  acid.  Cymene  is  oxidized  to  cumic  acid,  xylene  to  toluic  acid, 
methylpyridine  to  pyridine-carboxylic  acid  in  the  same  way.  If  the  side  chain 
has  several  members,  the  behavior  is  somewhat  different.  Phenylacetic  acid, 
C6H5.CH2.COOH,  in  which  only  one  carbon  atom  exists  between  the  ben- 
zene nucleus  and  the  carboxyl,  is  not  oxidized,  but  is  eliminated  after  con- 
jugation with  glycocoll  as  phenaceturic  acid  (Salkowski^).  Phenylamino- 
acetic  acid,  CeHs.CHNHo.COOH  is  in  part  converted  into  mandelic  acid 
(phenylglycollic  acid),  CeHs.CHOH.COOH,  and  in  great  part  is  eliminated 
as  such  (ScHOTTEN,  Knoop  •*) .  Phenylpropionic  acid^  C6H5.CH2.CH2.COOH, 
with  three  carbon  atoms  in  the  side  chain,  is,  on  the  contrary,  oxidized 
into  benzoic  acid,  and  H.  and  E.  Salkowski^  have  ]3roposed  the  rule  that 
the  homologues  of  the  benzoic  acids  are  converted  into  benzoic  acid  when 
the  side  chain  contains  more  than  two  carbon  atoms. 

Knoop  has  shown  by  experiments  with  several  acids,  such  as  phenyl- 
butyric  acid,  phenyl-a-lactic  acid  and  others  that  this  rule  does  not  hold 
good.  The  phenylbutyric  acid,  C6H5CH2.CH2.CH2.COOH,  is  not  oxidized 
in  the  animal  body  into  benzoic  acid,  but  into  phenylacetic  acid,  and  the 
phenyl-a-lactic  acid,  C6H5.CH2.CH(OH).COOH,  is  decomposed  nearly  en- 
tirely and  only  a  small  residue  is  eliminated  unchanged.  Knoop  has,  on  the 
contrary,  made  it  very  probable  that,  at  least  for  the  saturated,  normal  fatty 
acids  wnth  phenyl  substituted  at  the  end,  on  their  oxidation  they  follow 
the  rule  that  the  carboxyl  group  produced  from  the  body  stands  in  the  /3 
position  to  the  original  carboxyl.  This  explains,  for  example,  the  forma- 
tion of  phenylacetic  acid  from  phenylbutyric  acid,  and  benzoic  acid  from 
phenylvalerianic    acid,    C6H5.CH2.CH2.CH2.CH2.COOH,  for    in    the    last- 

'  Schultzen  and  Naunyn,  Reichert  and  Arch.  f.  (Anat.  u.)  Physiol.,  1867;  Baumann 
and  Preusse,  Zeitschr.  f.  physiol.  Chem.,  3,  156.  See  also  Nencki  and  Giacosa,  ibid.,  4; 
Lesnik  and  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  24;  F.  Miiller,  Deutsch.  med. 
Wochen.schr. ,  1S87;  Jaffe  and  Hilbert,  Zeitschr.  f.  physiol.  Chem.,  12;  Morner,  ibid., 
13;  Klingenberg,  "  Studien  iiber  die  Oxydation  aromatischer  Substanzen,"  etc. 
Inaug.-Diss.  Rostock,  1891.  In  regard  to  formanilid,  which  acts  essentially  as 
acetanilid,  see  Kleine,  Zeitschr.  f.  physiol.  Chem.,  22. 

'  Zeitschr.  f.  physiol.  Chem.,  4. 

''■Ibid..  7  and  9. 

*  Ibid.,  8. 

'Ibid.,  7. 


CASUAL   URINARY    CONSTITUENTS.  635 

■mentioned  case  phenylpropionic  acid  must  first  be  produced  and  then 
benzoic  acid  from  this.  Exceptions  to  this  rule  are  the  propionic  acids 
substituted  in  the  a-position,  i.e.,  phenylalanine,  phenyl-a-lactic  acid,  and 
phenyl-a-ketopropionic  acid,  which,  like  tyrosine  and  a-amino-cinnamic 
acid,  are  burnt  in  the  body.  Schotten's  rule,  according  to  which  all  acids 
having  three  carbon  atoms  in  the  side  chain  of  which  the  middle  one  has 
a  NH2  group  attached,  are  nearly  completely  burnt  in  the  organism,  has 
been  extended  by  these  exceptions. 

If  several  side  chains  are  present  in  the  benzene  nucleus,  then  only  one 
is  always  oxidized  into  carbox}^.  Thus  xylene,  CoR^iClis)-,,  is  oxidized 
into  toluic  acid,  C6H4(CH3)COOH  (Schultzen  and  Naunyn);  fuesitylene, 
C6H3(CH3)3.  into  mesitylenic  acid,  C6H3(CH3)2.COOH  (L.  Nencki);  cymene. 
(CH3)2CH.C6H4.CHo,  into  cumic  acid  (M.  Nencki  and  Ziegler^);    and 

vanillin,  OH.C6H3<pttqS  into,  vanilUnic  acid  (Y.  Kotake),^ 

Reductions  may  also  occur  and  examples  of  this  kind  are  the  conver- 
sion, as  observed  by  E.  MEYER,^of  nitrobenzene,  C6H5NO2,  or  ol  nitro phenol, 
HO.C6H4.NO2  into  aminophenol,  HO.C6H4.NH2,  and  also  the  behavior 
of  m-nitrobenzaldehyde  in  the  animal  body  as  mentioned  below. 

Syntheses  of  aromatic  substances  with  other  atomic  groups  occur  fre- 
quently. To  these  syntheses  belongs,  in  the  first  place,  the  conjugation  of 
benzoic  acid  with  glycocoll  to  form  hippuric  acid,  the  discovery  of  which  is 
generally  ascribed  to  Wohler,  but  according  to  Heffter  ■*  more  correctly 
to  Keller  and  Ure.  All  the  numerous  aromatic  substarices  which  are 
converted  into  benzoic  acid  in  the  body  are  voided  partly  as  hippuric  acid. 
This  statement  is  not  true  for  all  species  of  animals.  According  to  the 
observations  of  Jaffe^  benzoic  acid  does  not  pass  into  hippuric  acid  in 
birds,  but  into  another  nitrogenous  acid,  ornithuric  acid,  C19H20N2O4.  This 
acid  yields  as  splitting  products,  besides  benzoic  acid,  ornithine,  a  body 
which  has  been  spoken  of  on  page  97.  Not  only  are  the  oxybenzoic  ocids 
and  the  substituted  benzoic  acids  conjugated  with  glycocoll,  forming  corre- 
sponding hippuric  acids,  but  also  the  above-mentioned  acids,  toluic,  mesity- 
lenic, cumic,  and  phenylacetic  acids.  These  acids  are  voided  as  toluric, 
mesitylenuric,  cuminuric,  and  phenaceturic  acids. 

It  must  be  remarked  in  regard  to  the  oxybenzoic  acids  that  a  conju- 
gation with  glycocoll  has  been  shown  only  with  salicylic  and  p-oxybenzoic 

>  L.  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  1;  Nencki  and  Ziegler,  Ber.  d.  deutsch. 
chem.  Gesellsch.,  5.     See  also  O.  Jacobsen,  ibid.,  12. 

^  Zeitschr.  f.  physiol.  Chem.,  46. 

'Ibid.,  46. 

"  Die  Ausscheidung  korperfremder  Substanzen  im  Harn.  Ergebnisse  der  PiiysioJ.,  4, 
252. 

^  Ber,  d.  d.  chem.  Gesellsch.,  10  and  11, 


636  URINE. 

acid  (Bertagnini,  Baumann,  Herter,  and  others),  while  Baumann  and 
Herter  ^  find  it  only  very  probable  for  m-ox^iDenzoic  acid.  The  oxy- 
benzoic  acids  are  also  in  part  eliminated  as  conjugated  sulphuric  acids, 
which  is  especially  true  for  w-oxybenzoic  acid.  The  three  aminolienzoic 
acids,  according  to  the  experiments  of  Hildebrandt,  on  rabbits,  appeared 
at  least  in  part  unchanged  in  the  urine.  Salkowski  found,  as  was  later 
confirmed  by  R.  Cohn,^  that  ?^2-aminobenzoic  acid  passes  in  part  into 
uvaminobenzoic  acid,  H2N.CO.HN.C6H4.COOH.  It  is  also  in  part  elimi- 
nated as  aminohippuric  acid. 

The  behavior  of  the  halogen-substituted  compounds  of  toluene  varies 
in  different  animals  according  to  Hildebrandt's  experiments.  In  dogs 
they  are  converted  into  the  corresponding  substituted  hippuric  acid.  In 
rabbits  o-bromtoluene  is  completely  changed  to  hippuric  acid,  the  m-  and 
p-bromtoluene  only  partly.  The  three  chlortoluenes  are  converted  in  rab- 
bits into  the  corresponding  benzoic  acid  and  are  eUminated  as  such  and 
not  as  hippuric  acid. 

The  substituted  aldehydes  are  of  special  interest  as  substances  which 
may  undergo  conjugation  with  glycocoU.  According  to  the  investigations 
of  R.  CoHN  3  on  this  subject  o-nitrohenzaldehyde  when  introduced  into  a 
rabbit  is  only  in  a  verj^  small  part  converted  into  nitrobenzoic  acid,  and 
the  chief  mass,  about  90  per  cent,  is  destroyed  in  the  body.  According 
to  SiEBER  and  Smirnow*  m-mitrobenzaldehyde  passes  in  dogs  into  m-nitro- 
hippuric  acid,  and  according  to  Cohn  into  urea-m.-nitrohippurate.  In 
rabbits  the  behavior  is  quite  different.  In  this  case  not  only  does  an  oxida- 
tion of  the  aldehyde  into  benzoic  acid  take  place,  but  the  nitro-group  is 
also  reduced  to  an  amino-group,  and  finally  acetic  acid  attaches  itseK  to 
this  with  the  expulsion  of  water,  so  that  the  final  product  is  m-acetyl- 
aminobenzoic  acid,  CH3.CO.NH.C6H4.COOH.  This  process  is  analogous 
to  the  beha-vior  of  furfurol,  and  the  reduction  does  not  take  place  in  the 
intestine,  but  in  the  tissues.  The  2?-nitrobenzaldehyde  acts  in  rabbits  in 
part  like  the  m-aldehyde  and  passes  in  part  into  p-acetylaminohenzoic  acid. 
Another  part  is  converted  into  p-nitrobenzoic  acid,  and  the  urine  contains 
a  chemical  combination  of  equal  parts  of  these  two  acids.  According  to 
SiEBER  and  Smirnow^  2>nitrobenzaldehyde  yields  only  urea  p-nitrohip- 
purate  in  dogs.     The  above-mentioned  pyridine-carhoxylic  acid,  formed  from 


'  Zeitschr.  f.  physiol.  Chem.,  1,  where  Bertagnini's  work  is  also  cited.  See  also 
Dautzenberg,  Maly's  Jahresber.,  11,  231. 

-Salkowski,  Zeitschr.  f.  physiol.  Chem.,  7;  Cohn,  ibid.,  17;  Hildebrandt,  Hof- 
meister's  Beitrage,  3. 

^  Zeitschr.  f.  physiol.  Chem.,  17. 

*  Monatshefte  f.  Chem.,  8. 


CASUAL   URINARY   CONSTITUENTS.  637 

methy [pyridine  (a-picoline)  passes  into  the  urine  after  conjugation  with 
glycocoll  as  oi-pyridinuric  acid} 

To  those  substances  which  undergo  a  conjugation  with  glycocoll  Ijelongs 
also  furjurol  (the  aldehyde  of  pyromucic  acid),  which,  when  introduced  into 
rabbits  and  dogs,  as  shown  by  Jaffe  and  Cohx.2  is  fust  oxidized  into  pyro- 
mucic acid  and  then  eUminated  as  pyromucuric  acid.  C7H7X4O,  after 
conjugation  with  glycocoll.  In  birds  this  behavior  is  different,  namely, 
the  acid  is  conjugated  with  another  substance,  ornithine,  C5Hi2N202,  which 
is  a  diamino valerianic  acid,  forming  pyromucinornithuric  acid. 

Furfurol  also  undergoes  conjugation  with  glycocoll  in  other  forms  in 
mammals.  Thus  Jaffe  and  Cohn  found  that  it  is  in  part  combined  with 
acetic  acid,  forming  furfuracrylic  acid,  C4H30.CH:CH.COOH,  which  passes 
into  the  urine  coupled  with  glycocoll  as  furjuracryluric  acid. 

It  has  not  been  proved  how  thiophene,  C4H4S,  behaves  in  the  animal 
body.  Of  methylthiophene  (thiotolene),  C4H3S.CH3,  a  very  small  part  is 
oxidized  to  thiophenic  acid,  C4H3S.COOH  (Levy).  This  acid,  as  shown 
by  Jaffe  and  Le\^,^  is  conjugated  with  glycocoll  in  the  body  (rabbits) 
and  eUminated  as  thiophenuric  acid. 

Another  ver}^  important  synthesis  of  aromatic  substances  is  that  of 
the  ethereal-sulphuric  acids.  Phenols  and  chiefly  the  hydroxylated  aromatic 
hydrocarbons  and  their  derivatives  are  voided  as  ethereal-sulphuric  acids, 
according  to  Baumann,  Herter  and  others.^ 

A  conjugation  of  aromatic  acids  with  sulphuric  acid  occurs  less  often. 
The  two  pre\aously-mentioned  aromatic  acids,  p-oxyphenylacetic  and  p-oxy- 
phenylpropionic  acid,  are  in  part  eliminated  in  this  form.  Gentisic  acid 
(hydroquinone-carboxylic  acid)  also  increases,  according  to  Likhatscheff.s 
the  quantity  of  ethereal-sulphuric  acid  in  the  urine,  and  according  to  RosT 
the  same  occurs,  contrarv-  to  the  older  statements,  with  gallic  acid  (trioxy- 
benzoic  acid)  and  tannic  acid.^ 

While  acetophenone  (phenylmethylketone) .  C6H5.CO.CH3,  as  shown  by 
M.  Nexcki,  is  oxidized  to  benzoic  acid  and  eliminated  as  hippuric  acid, 
the  aromatic  oxyketones  with  hydroxjd   gTOups,  such  as  resacetophenone, 

*  In  regard  to  the  extensive  literature  on  glycocoll  conjugations  we  refer  the  reader 
to  O.  Kiihling,  Ueber  Stoffwechselprodukte  aromatischer  Korper.  Inaug.-Diss., 
Berlin,  1SS7. 

2  Ber.  d.  d.  Chem.  Gesellsch  ,  20  and  21. 

^  Le\'y,  Ueber  das  Verlialten  einiger  Thiophenderivate,  etc.,  Inaug.-Diss.,  Konigs- 
berg,  1889;    Jaffe  and  Levy,  Ber.  d.  d.  chem.  Gesellsch.,  21. 

*  In  regard  to  the  hterature,  see  O.  Kiihling,  1.  c. 
^Zeitschr.  f.  physiol.  Chem.,  21. 

*  In  regard  to  the  behavior  of  gallic  and  tarmic  acids  in  the  animal  body,  see  C. 
Momer,  Zeitschr.  f.  physiol.  Chem.,  16,  wliich  also  contains  the  older  literature;  also 
Harnack,  ibid.,  21,  and  Rost,  Arch.  f.  exp.  Path.  u.  Pharm.,  3S,  and  Sitzungsber.  d. 
Gesellsch.  zur  Beford.  d   ges.  Naturmss.  201  Marburg,  1898. 


638  URINE. 

G6H3(OH)(OH)(CO.CH3),   paraoxijpropiophenone,   C6H4(OH)(COCH2.CH3), 

12  3  4 

and  gallacetophenone,  C6H2(OH)(OH)(OH)(CO.CH3),  pass  into  the  urine 
without  previous  oxidation  as  ethereal-sulphuric  acids  and  in  part  after 
conjugation  with  glucuronic  acid  (Nexcki  and  RekoW'SKI^).  Euxanthon, 
which  is  also  an  aromatic  oxyketone,  passes  into  the  urine  as  euxanthic  acid 
after  the  conjugation  with  glucuronic  acid  previously  mentioned. 

A  conjugation  of  other  aromatic  substances  with  glucuronic  acid ,  wliich 
last  is  protected  from  combustion,  occurs  rather  often.  The  phenols,  as 
above  stated  (page  590),  pass  in  part  as  conjugated  glucuronic  acids  into 
the  urine.  The  same  is  true  for  the  homologues  of  the  phenols,  for  certain 
substituted  phenols,  and  for  many  aromatic  substances,  also  hydrocarbons 
after  pre\dous  oxidation  and  hj'dration.  Thus  Hildebr.\ndt  and  Fromm 
and  Clemens  -  have  shown  that  the  cyclic  terpenes  and  campfiors,  by  oxida- 
tion or  hydration,  or  in  certain  cases  by  both,  are  converted  into  hydroxyl 
derivatives  when  the  body  in  question  is  not  previous^  hydroxylized,  and 
that  these  hydroxyl  derivatives  are  eliminated  as  conjugated  glucuronic 
acids.  Conjugated  glucuronic  acids  are  detected  in  the  urine  after  the  intro- 
duction of  various  substances,  e.g.,  therapeutic  agents  into  the  organism, 
namely,  terpenes,  horneol,  menthol,  camphor  (camphoglucuronic  acid  was 
first  observed  by  Schmiedeberg),  naphtJialene,  oil  of  turpentine,  oxyquino- 
lines,  antipyrine,  and  many  other  bodies.^  Orthonitrotoluene  in  dogs  passes 
first  into  o-nitrobenzyl  alcohol  and  then  into  a  conjugated  glucuronic  acid, 
uronitrotoluolic  acid  (Jaffe).*  The  glucuronic  acid  split  off  from  this 
conjugated  acid  is  levogyrate  and  hence  is  not  identical  but  only  isomeric 
wdth  the  ordinary  glucuronic  acid.  Ditnethylaminohenzaldehyde,  according 
to  Jaffe,  is  converted  in  part  into  dimethylaminobenzoglucuronic  acid 
in  rabbits.  The  same  conjugated  glucuronic  acid  is  also  produced,  accord- 
ing to  HiLDEBRAXDT,5  from  p-dimethyltoluidine ,  which  is  first  changed  into 
p-dimeihylaminohenzoic  acid.  Indol  and  skatol  seem,  as  above  stated 
(page  595),  to  be  eUminated  in  the  urine  partly  as  conjugated  glucuronic 
acids. 

A  synthesis  in  which  compounds  containing  sulphur,  iJiercapturic  acids,  are 
formed  and  eliminated  after  conjugation  with  glucuronic  acid,  occurs  when 

'  Arch.  d.  scienc.  bid.  de  St.  Petersbourg,  3,  and  Ber.  d.  deutsch.  chem.  Gesell- 
sch.,  27. 

'  Hildebrandt,  Arch.  f.  exp.  Path.  u.  Pharm.,  45,  46;  Zeitschr.  f.  physiol.  Chem., 
36;  with  Fromm,  ib/d.,  33;  and  with  Clemens,  ibid.,  37;  Fromm  and  Clemens, /6id.,  34. 

^  See  O.  Kiihling,  1.  c,  which  gives  the  literature  up  to  1887;  also  E.  Kiilz,  Zeitsclir. 
f.  Biologic,  27;  the  works  of  Hildebrandt,  Fromm  and  Clemens,  see  foot-note,  2; 
Brahm,  Zeitschr.  f.  physiol.  Chem.,  28;  Fen}^-essy,  ibid.,  30;  Bonanni,  Hofmeister's 
Beitrage,  1;   Lawrow,  Ber.  d.  d.  chem.  Gesellsch.,  33. 

■•  Zeitschr.  f.  physiol.  Chem.,  2. 

'Jaffe,  Zeitschr.  f.  physiol.  Chem.,  43;    Hildebrandt,  Hofmeister's  Beitrage,  7. 


CASUAL   URINARY  CONSTITUENTS.  639 

chlorine  and  bromine  derivatives  of  benzene  are  introduced  into  the  organism 
of  dogs  (Bau]vl^*x  and  Pretjsse,  Jaffe).  Thus  chlorhenzene  combines 
with  cysteine,  forming  chlorphenylmercapturic  acid,  C11H12CISXO3.  The 
important  inxestigations  of  FRIED^L\xx  ^ show  that  the phen3-lthiolactic  acid 
which  forms  the  foundation  of  the  mercapturic  acids  belongs  to  the  .j-series, 
and  in  this  way  the  direct  chemical  connection  of  this  body  with  the  pro- 
tein-cystine  (a:-amino-^5-thiolactic  acid)  is  established.  Fried^lvnx  has 
also  been  able  to  convert  cysteine  into  bromphenylmercapturic  acid. 

Pyridine,  C5H5X,  which  does  not  combine  either  with  glucuronic  acid 
or  with  sulphuric  acid  after  pre\ious  oxidation,  shows  a  special  beha\dor. 
It  takes  up  a  methyl  group  as  found  by  His  and  later  confirmed  by  Cohn  ^ 
and  forms  an  ammonium  combination,  methylpyridylammonium  hydroxide, 
HO.CH3.XC5H5. 

Several  alkaloids,  such  as  quinine,  morphine,  and  strychnine,  may  pass 
into  the  urine.  After  the  ingestion  of  turpentine,  balsam  of  copaiva,  and 
resins,  these  may  appear  in  the  urine  as  resin  acids.  Different  kinds  of 
coloring-matters,  such  as  alizarin,  crysophanic  acid,  after  rhubarb  or  senna, 
and  the  coloring-matter  of  the  blueberry,  etc.,  may  also  pass  into  the  urine. 
After  rhubarb,  senna,  or  santonine  the  urine  assumes  a  yellow  or  greenish- 
yellow  color,  which  is  transformed  into  a  beautiful  red  by  the  addition 
of  alkali.  Phenol  produces,  as  above  mentioned,  a  dark-brown  or  dark- 
green  color  which  depends  mainly  on  the  decomposition  products  of  hydro- 
quinone  and  humin  substances.  After  naphthalene  the  urine  has  a  dark 
color,  and  several  other  medicinal  agents  produce  a  special  coloration. 
Thus  after  antipyrine  it  becomes  yellow  or  blood-red.  After  balsam  of 
copaiva  the  urine  becomes,  when  strongly  acidified  with  hydrochloric  acid, 
gradually  rose-  and  purple-red.  After  naphthalene  or  naphthol  the  urine 
gives  with  concentrated  sulphuric  acid  (1  c.c.  of  concentrated  acid  and  a 
few  drops  of  urine)  a  beautiful  emerald-green  color,  which  is  probably  due 
to  naphthol-glucuronic  acid.  Odoriferous  bodies  also  pass  into  the  urine. 
After  asparagus  the  urine  acquires  a  disgusting  odor  which  is  probably  due 
to  methylmercaptan,  according  to  ^^I.  Xexcki.^  After  turpentine  the  urine 
may  have  a  pecuUar  odor  similar  to  that  of  \dolets. 

*  Baumann  and  Preusse,  Zeitschr.  f.  physiol.  Chem.,  5;  Jaflfe,  Ber.  d.  deutsch. 
chem.  Gesellsch.,  12;   Friedmann,  Hofmeister's  Beitrage,  4. 

-  His,  Arch  f .  exp.  Path.  u.  Pharm.,  22;  Cohn,  Zeitschr.  f.  physiol.  Chem.,  18. 
^  Arch.  f.  exp.  Path.  u.  Pharm.,  28. 


640  URINE. 


VI.    Pathological  Constituents  of  Urine. 

Proteid.  The  appearance  of  slight  traces  of  proteid  in  normal  urines 
has  been  repeatedly  observed  by  many  investigators,  such  as  Posnee, 
Plosz.  v.  Noorden.  Leube,  and  others.  According  to  K.  ^Iorxer  ^  pro- 
teid regularly  occui-s  as  a  normal  urinary  constituent  to  the  extent  of  22-78 
milligrams  per  liter.  Frequently  traces  of  a  substance  similar  to  a  nucleo- 
albumin,  which  is  easily  mistaken  for  mucin,  and  whose  nature  will  be 
treated  of  later,  appear  in  the  urine.  In  diseased  conditions  proteid 
occurs  in  the  urine  in  a  variety  of  cases.  The  albuminous  bodies  wliich 
most  often  occur  are  serglobulin  and  seralbumin.  Proteoses  (or  pep- 
tones) are  also  sometimes  present.  The  quantity  of  proteid  in  the  urine 
is  in  most  cases  less  than  5  p.  m.,  rarely  10  p.  m.,  and  only  very  rarely  does 
it  amount  to  50  p.  m.  or  over.  Cases  are  known,  however,  where  it  was 
even  more  than  80.  p  m. 

Among  the  many  reactions  proposed  for  the  detection  of  proteid  in 
urine,  the  following  are  to  be  recommended: 

The  Heat  Test.  Filter  the  urine  and  test  its  reaction.  An  acid  urine 
may,  as  a  rule,  be  boiled  without  further  treatment,  and  only  in  especially 
acid  urines  is  it  necessary'  to  first  treat  with  a  little  alkali.  An  alkaline 
urine  is  made  neutral  or  faintly  acid  before  heating.  If  the  urine  is  poor  in 
salts,  add  1-10  vol.  of  a  saturated  common-salt  solution  before  boiling;  then 
heat  to  the  boiling-point,  and  if  no  precipitation,  cloudiness,  or  opalescence 
appears,  the  urine  in  question  contains  no  coagulable  proteid,  but  it  may 
contain  proteoses  or  peptones.  If  a  precipitate  is  produced  on  boiUng,  this 
may  consist  of  proteid,  or  of  earthy  phosphates,  or  of  both.  The  mono- 
hydrogen  calcium  phosphate  decomposes  on  boiling,  and  the  normal  phos- 
phate may  separate  out.  The  proper  amount  of  acid  is  now  added  to  the 
urine,  so  as  to  prevent  any  mistake  caused  by  the  presence  of  earthy  phos- 
phates, and  to  give  a  better  and  more  flocculent  precipitate  of  the  proteid. 
If  acetic  acid  is  used  for  this,  then  add  1-3  drops  of  a  25  per  cent  acid 
to  each  10  c.c.  of  the  urine  and  boil  after  the  addition  of  each  drop.  On 
using  nitric  acid,  add  1-2  drops  of  the  25  per  cent  acid  to  each  cubic  centi- 
meter of  the  boiling-hot  urine. 

On  using  acetic  acid,  when  the  quantity  of  proteid  is  very  small,  and 
especiallv  when  the  urine  was  originally  alkaline,  the  proteid  may  some- 
times remain  in  solution  on  the  addition  of  the  above  quantity  of  acid. 
If,  on  the  contrar}^  less  acid  is  added,  the  precipitate  of  calcium  phosphate, 
which  forms  in  amphoteric  or  faintly  acid  urines,  is  liable  not  to  dissolve 
completely,  and  this  may  cause  it  to  be  mistaken  for  a  proteid  precipitate. 
If  nitric  acid  is  used  for  the  heat  test,  the  fact  must  not  be  overlooked  that 
after  the  addition  of  only  a  little  acid  a  combination  between  it  and  the 
proteid  is  formed  which  is  soluljle  on  boiling  and  which  is  only  precipitated 
by  an  excess  of  the  acid.  On  this  account  the  large  quantity  of  nitric  acid, 
as  suggested  above^  must  be  added,  but  in  this  case  a  small  part  of  the 

*  Skand.  Arch.  f.  Physiol.,  6  (hteiature). 


PROTEID.  641 

proteid  is  liable  to  be  dissolved  by  the  excess  of  the  nitric  acid-  When 
the  acid  is  added  after  boiling,  which  is  absolutely  necessary,  the  liability 
of  a  mistake  is  not  so  great.  It  ib  on  these  grounds  that  the  heat  test, 
although  it  gives  very  good  results  in  the  hands  of  experts,  is  not  recom- 
mended to  physicians  as  a  positive  test  for  proteid. 

A  confounding  with  mucin,  when  this  body  occurs  in  the  urine,  is  easily 
prevented  in  the  heat  test  with  acetic  acid  by  acidifj-ing  another  portion 
with  acetic  acid  at  the  ordinary'  temj^jerature.  Mucin  and  nucleoalbumin 
substances  similar  to  mucin  are  hereby  precipitated.  If  in  the  perform- 
ance of  the  heat  and  nitric -acid  test  a  precipitate  fu'st  appears  on  cooling 
or  is  strikingly  increased,  then  this  shows  the  presence  of  proteoses  in  the 
urine,  either  alone  or  mixed  with  coagulable  proteid.  In  this  case  a  further 
investigation  is  necessary  (see  telow).  In  a  urine  rich  in  urates  a  precipitate 
consisting  of  uric  acid  separates  on  cooling.  This  precipitate  is  colored  and 
granular,  and  is  hardly  to  be  mistaken  for  an  proteoses  or  proteid  precipitate. 

Heller's  test  is  performed  as  follows  (see  page  41) :  The  urine  is  ver}' 
carefully  floated  on  the  surface  of  nitric  acid  in  a  test-tube.  The  presence 
of  proteid  is  shown  by  a  white  ring  between  the  two  liquids.  ^^  ith  this 
test  a  red  or  reddish-violet  transparent  ring  is  always  obtained  ■uith  normal 
urine;  it  depends  upon  the  indigo  coloring-matters  and  can  hardly  be  mis- 
taken for  the  white  or  whitish  proteid  ring,  and  this  last  must  not  be  mis- 
taken for  the  ring  produced  by  bile-pigments.  In  a  urine  rich  in  urates 
another  complication  may  occur,  due  to  the  formation  of  a  ring  produced 
by  the  precipitation  of  uric  acid.  The  uric-acid  ring  does  not  lie,  like  the 
proteid  ring,  between  the  two  liquids,  but  somewhat  higher.  For  this  rea- 
son two  simultaneous  rings  may  exist  in  urines  which  are  rich  in  urates  and 
do  not  contain  ver}^  much  proteid.  The  disturbance  caused  by  uric  acid 
is  easily  prevented  by  diluting  the  urine  with  1-2  vols,  of  water  before 
performing  the  test.  The  uric  acid  now  remains  in  solution,  and  the  deli- 
cacy of  Heller's  test  is  so  great  that  after  dilution  only  in  the  presence 
of  insignificant  traces  of  proteid  does  this  test  give  negative  results.  In 
a  urine  ver\^  rich  in  urea  a  ring-like  separation  of  urea  nitrate  may  also 
appear.  This  ring  consists  of  shining  cr^'stals,  and  it  does  not  appear 
in  urine  pre\'iously  diluted.  A  confusion  with  resinous  acids,  Avhich  also 
give  a  whitish  ring  with  this  test,  is  easily  prevented,  since  these  acids 
are  soluble  on  the  addition  of  ether.  Stir,  add  ether,  and  carefully  shake 
the  contents  of  the  test-tube.  If  the  cloudiness  is  due  to  resinous  acids, 
the  urine  gradually  becomes  clear,  and  on  evaporating  the  ether  a  sticky 
residue  of  resinous  acids  is  obtained.  A  liquid  which  contaias  tme  mucin 
does  not  give  a  precipitate  with  this  test,  but  it  gives  a  more  or  less  strongly 
opalescent  ring,  which  disappears  on  stirring.  The  liquid  does  not  con- 
tain any  precipitate  after  stirring,  but  is  somewhat  opalescent.  If  a  faint, 
not  wholly  typical  reaction  is  obtained  with  Heller's  test  after  some 
time  with  undiluted  urine,  while  the  diluted  urine  gives  a  pronounced 
reaction,  the  presence  is  shown  of  the  substance  which  used  to  be  called 
mucin  or  nucleoalbumin.  In  this  case  proceed  as  described  below  for  the 
detection  of  nucleoalbumin. 

If  the  above-mentioned  possible  errors  and  the  means  by  which  they  may 
be  prevented  are  borne  in  mind,  there  is  hardly  another  test  for  proteid 
in  the  urine  which  is  at  the  same  time  so  easily  performed,  so  delicate,  and 
so  positive  as  Heller's.  With  this  test  even  0.002  per  cent  ff  albumin 
may  be  detected  without  difficulty.     Still  the  student  must  not  be  satisfied 


642  URINE. 

with  this  test  alone,  but  should  apply  at  least  a  second  one,  such  as  the  heat 
test.     In  performing  this  test  the  (primary)  proteoses  are  also  precipitated. 

The  reaction  with  meta phosphoric  acid  (see  page  41)  is  very  convenient 
and  easily  performed.  It  is  not  quite  so  delicate  and  positive  as  Helllr's 
test.     The  proteoses  are  also  precipitated  by  this  reagent. 

Reaction  with  Acetic  Acid  and  Potasstiun  Ferrocyanide.  Treat  the  urine 
first  with  acetic  acid  until  it  contains  about  2  per  cent,  and  then  add  drop 
by  drop  a  potassium-ferrocyanide  solution  (1:20),  carefully  avoiding  an 
excess.  This  test  is  very  good,  and  in  the  hands  of  experts  it  is  even  more 
delicate  than  Heller's".  In  the  presence  of  ver}-  small  quantities  of  pro- 
teid  it  requires  more  practice  and  dexterity  than  Heller's,  as  the  relative 
quantities  of  reagent,  proteid,  and  acetic  acid  influence  the  result  of  the 
test.  The  quantity  of  salts  in  the  urine  likewise  seems  to  have  an  infiuence. 
This  reagent  also  precipitates  proteoses. 

Spiegler*s  Test.  Spiegler  recommends  a  solution  of  8  parts  mercuric 
chloride,  4  parts  tartaric  acid,  20  parts  gl3^cerine,  and  200  parts  water  as  a  very 
delicate  reagent  for  proteid  in  the  urine.  A  test-tube  is  half  filled  with  this- 
reagent  and  from  a  pipette  the  urine  is  allowed  to  flow  upon  its  surface  drop  by 
drop  along  the  wall  of  the  test-tube.  In  the  presence  of  proteid  a  white  ring  is 
obtained  at  the  point  of  contact  between  the  two  liquids.  The  delicacy  of  this 
test  is  1:3.50,000.  .Jolles  '  does  not  consider  this  reagent  suited  for  urines  very 
poor  in  chlorine,  and  for  this  reason  he  has  changed  it  as  follows:  10  grams  mer- 
curic chloride,  20  grams  succinic  acid,  10  grams  XaCl,  and  500  c.c.  water. 

Rocpi's  Test.  Treat  the  urine  either  with  a  20  per  cent  watery  solution  of 
sulphosalicylic  acid  or  a  few  crystals  of  the  acid.  This  reagent  does  not  pre- 
cipitate the  uric  acid  or  the  resin  acids.^ 

As  every  normal  urine  contains  traces  of  proteid,  it  is  apparent  that 
very  dehcate  reagents  are  to  be  used  only  with  the  greatest  caution.  For 
ordinary  cases  Heller's  test  is  sufficiently  delicate.  If  no  reaction  Is 
obtained  with  this  test  within  2^  to  3  minutes,  the  urine  tested  contains 
less  than  0.003  per  cent  of  proteid,  and  is  to  be  considered  free  from  proteid 
in  the  ordinary'  sen.?e. 

The  use  of  precipitating  reagents  presumes  that  the  urine  to  be  investi- 
gated is  perfectly  clear,  especially  in  the  presence  of  only  very  little  pro- 
teid. The  urine  must  first  be  filtered.  This  is  not  easily  done  with  urine 
containing  bacteria,  but  a  clear  urine  may  be  obtained,  as  suggested  by 
A.  Jolles,  by  shaking  the  urine  with  infusorial  earth.  Although  a  little 
proteid  is  retained  in  this  procedure  and  lost,  H  does  not  seem  to  be  of  any 
importance  (Grutzxer.  Schw'eissinger^). 

The  different  color  reactions  cannot  be  directly  used,  especially  in  deep- 
colored  urines  which  contain  only  little  proteitl.  The  common  salt  of  the 
urine  has  a  disturbing  action  on  ^Iillon's  reagent.  To  prove  more  posi- 
tively the  presence  of  proteid,  the  precipitate  ol^tained  in  the  boiling  test 

'  Spiegler.  Wien.  klhi.  Wochenschr.,  1S92,  and  Centralbl.  f.  d.  klin.  Med.,  1S93; 
Jolles.  Zeitschr.  f.  physiol.  Chem.,  21. 

^  Pliarmaceut.  Centralbl.,  1889,  and  Zeitschr.  f.  phy.-iol.  Chem.,  29. 

^  .Ifdles,  Zeitschr.  f.  anal.  Chem.,  21);  CJriitzner,  Chem.  Cenitalbl.,  1901,  1;  Schweis- 
sineer.  ibid. 


DETECTION    OF   GLOBULIN    AND    ALBUMIN.  643 

may  be  filtered,  washed,  and  then  tested  with  Millox's  reagent.  The 
precipitate  may  also  be  dissolved  in  dilute  alkali  and  the  biuret  test  applied 
to  the  solution.  The  presence  of  proteoses  or  peptones  in  the  urine  is 
directly  tested  for  by  this  last-mentioned  test.  In  testing  the  urine  for 
proteid  one  should  never  be  satisfied  with  one  reaction  alone,  but  must 
apply  the  heat  test  and  Heller's  or  the  potassium-ferrocyanide  test.  In 
using  the  heat  test  alone  the  proteoses  may  be  easily  overlooked,  but  these 
are  detected,  on  the  contrar}-,  by  Heller's  or  the  potassium-ferrocyanide 
test.  If  only  one  of  these  tests  is  employed,  no  sufficient  intimation  of  the 
kind  of  proteid  present  can  be  obtained,  whether  it  consists  of  proteoses 
or  coagulable  proteid. 

For  practical  purposes  several  dry  reagents  for  proteid  have  been  recommended. 
Besides  the  metaphosphoric  acid  may  be  mentioned  Stutz's  or  Furbrixger's 
gelatine  capsules,  which  contain  mercuric  cUoride,  sodium  chloride,  and  citric 
acid;  and  Geissler's  albumin-test  papers,  which  consist  of  strips  of  filter-paper 
which  have  been  dipped  in  a  solution  of  citric  acid  and  also  mercuric-chloride  and 
potassium-iodide  solution  and  then  dried. 

If  the  presence  of  proteid  has  been  positively  proved  in  the  urine  by 
the  above  tests,  it  then  remains  necessarj^  to  determine  its  character. 

The  Detection  of  Globulin  and  Albumin.  In  detecting  serglobuUn  the 
urine  is  exactly  neutralized,  filtered,  and  treated  with  magnesium  sulphate 
in  substance  until  it  is  completely  saturated  at  the  ordinary  temperature, 
or  with  an  equal  volume  of  a  saturated  neutral  solution  of  ammonium  sul- 
phate. In  both  cases  a  white,  flocculent  precipitate  is  formed  in  the 
presence  of  glubulin.  In  using  ammonium  sulphate  with  a  urine  rich  in 
urates  a  precipitate  consisting  of  ammonium  urate  may  separate.  This 
precipitate  does  not  appear  immediately,  but  only  after  a  certain  time,  and 
it  must  not  be  mistaken  for  the  globulin  precipitate.  In  detecting  ser- 
albumin heat  the  filtrate  from  the  globuUn  precipitate  to  boihng-point,  or 
add  about  1  per  cent  acetic  acid  to  it  at  the  ordinary  temperature. 

For  the  detection  and  also  for  the  quantitative  estimation  of  the  various 
globulins  (fibringlobulin,  euglobulin,  and  pseudoglobulin)  Oswald  '  has  proposed 
the  fractional  precipitation  with  ammonium  sulphate.  It  is  still  a  question 
whether  this  method,  which  is  not  quite  reliable,  can  be  used  in  urine  investi- 
gations. 

Proteoses  and  peptones  have  been  repeatedly  found  in  the  urine  in 
different  diseases.  ReUable  reports  are  at  hand  on  the  occurrence  of  pro- 
teoses in  the  urine.  The  statements  in  regard  to  the  occurrence  of  pep- 
tones date  from  a  time  when  the  conception  of  proteoses  and  peptones 
was  different  from  that  of  the  present  day,  and  in  part  they  are  based  upon 
investigations    using  untrustworthy    methods.      According    to    Ito  -    true 

^  Miinch.  med.  VVochenschr.,  1904. 

^  In  regard  to  the  literature  on  proteoses  and  peptones  in  urine,  see  Huijpert- 
Neubauer,  Ham-Analyse,  10.  Aufl.,  466  to  492;  also  A.  Stoffregen,  Ueber  das  Vorkom- 
nien  von  Pepton  im  Ham,  Sputum  und  Eiter  (Inaug.-Diss.,  Dorpat,  1891);  E.  Hirsch- 
feldt,  Ein  Beitrag  zur  Frags  der  Peptonurie  (Inaug.-Diss.,  Dorpat,  1892);  and  espe- 
cially Stadelmann.  Urtersuchungen  iiberdie  Peptonurie.  Wiesbaden,  1894;  Ehrstrom^ 
Bidrag  till  kannedomen  om  Albumosurien,  Helsingfors,  1900;  Ito,  Deutsch.  Arch, 
f.  klin.  Med.,  71. 


644  URINE. 

peptones  are  sometimes  found  in  the  urine  in  cases  of  pneumonia;  what 
has  been  designated  as  urine  peptone  seems  to  have  been  chiefly  deutero- 
peptones. 

In  detecting  the  proteoses  the  proteid-free  urine,  or  urine  boiled  with 
addition  of  acetic  acid,  is  saturated  with  ammonium  sulphate,  which  precipi- 
tates the  proteoses.  Several  errors  are  here  possible.  The  urobilin,  which 
may  give  a  reaction  similar  to  the  biuret  reaction,  is  also  precipitated  and 
may  lead  to  mistakes  (Salkow^ski,  Stokvis  i).  A  small  quantity  of  the 
proteid  may  remain  in  solution  after  coagulation  and  tliis  may  be  precipi- 
tated by  the  ammonium  sulphate  and  be  mistaken  for  proteoses.  The 
coagulable  proteid  may  be  completely  precipitated  by  saturating  with 
ammonium  sulphate  in  boiling  solution;  but  according  to  Devoto^  small 
quantities  of  proteose  may  be  formed  from  the  proteid  by  heating  for  a  long 
time  with  the  salt.  On  heating  for  a  short  time  no  such  formation  of 
proteose  takes  place,  and  the  proteids  are  completely  coagiilated. 

For  these  reasons  Bang^  has  suggested  the  following  method  for  the 
■detection  of  proteoses  in  the  presence  of  coagulable  proteid.  The  urine  is 
heated  to  boiling  with  ammonium  sulphate  (8  parts  to  10  parts  urine) 
and  boiled  for  a  few  seconds.  The  hot  liquid  is  centrifuged  for  ^  to  1  min- 
ute and  separated  from  the  sediment.  The  urobilin  is  removed  from 
this  by  extraction  \^dth  alcohol.  The  residue  is  suspended  in  a  Uttle  water, 
heated  to  boiling,  filtered,  whereby  the  coagulable  proteid  is  retained 
on  the  filter,  and  any  urolDilin  still  present  in  the  filtrate  is  shaken  out 
with  chloroform.  The  watery  solution,  after  removal  of  the  chloroform, 
is  used  for  the  biuret  test.  For  clinical  purposes  this  method  is  very  ser- 
A^iceable. 

According  to  Salkowski  the  urine  treated  with  10  per  cent  hydrochloric 
acid  is  precipitated  with  phosphotungstic  acid,  then  warmed,  the  liquid 
decanted  from  the  resin-like  precipitate,  this  washed  with  water,  and 
then  dissolved  in  a  little  water  with  the  aid  of  some  caustic  soda,  wanned 
again  until  the  blue  color  disappears,  cooled,  and  finally  tested  with  copper 
£!ulphate.  This  method  has  been  recently  somewhat  modified  by  V.  Aldor 
and  Cerny.'*  In  regard  to  other  more  complicated  methods  we  refer  to 
Huppert-Neubauer. 

.MoRAWiTZ  and  Dietschy  ^  first  remove  the  proteid  from  the  urine  made 
faintly  acid  with  acid  potassium  phosphate  by  the  addition  of  double  the 
volume  of  96  per  cent  alcohol  and  warming  on  the  water-bath  for  several 
hours.  From  the  concentrated  filtrate  acidified  with  a  little  sulphuric 
acid  the  proteoses  can  be  precipitated  by  saturating  with  zinc  sulphate. 
After  the  re»moval  of  the  urobilin  by  alcohol  and  extracting  with  water,  the 
biuret  test  may  be  applied. 

If  the  proteoses  have  been  precipitated  from  a  larger  portion  of  urine 
by  ammonium  sulphate,  this  precipitate  is  tested  for  the  presence  of  dif- 
ferent proteoses  for  the  reasons  given  in  Chapter  II.     The  following  serves 

'Salkowski,  Berlin,  klin.  Wochenschr.,  1897;   Stokvis,  Zeitschr.  f.  Biologie,  34. 
^  Zeitschr   f.  physiol.  Chem  ,  15. 
^  Deutsch   med.  Wochenschr.,  1898. 

••  Salkowski,  Centralbl.  f.d.  med.  Wissensch.,  1894;  v  Aldor,  Berl.  klin.  Wochenschr., 
S6;   6erny,  Zeitschr.  f.  analyt.  Chem.,  40. 
*Arch.  {.  exp.  Path.  u.  Pharm.,  'yi. 


ESTIMATION    OF    PROTEID    L\    URIXE.  645 

as  a  preliminar}'  determination  of  the  character  of  the  proteoses  present 
in  the  urine.  If  the  urine  contains  only  deuteroproteose  it  does  not  become 
cloudy  on  boiling,  does  not  give  Heller's  test,  does  not  become  cloudy 
on  saturating  with  NaCl  in  neutral  reaction,  but  does  become  turbid  on 
adding  acetic  acid  saturated  with  tliis  salt.  In  the  presence  of  onl}-  proto- 
proteose  the  urine  gives  Heller's  test,  is  precipitated  even  in  neutral 
solution  on  saturating  with  NaCl,  but  does  not  coagulate  on  boiling.  Tlie 
presence  of  heteroproteose  is  shown  by  the  urine  behaving  like  the  above 
with  NaCl  and  nitric  acid,  but  shows  a  difTerence  on  heating.  It  gradually 
becomes  cloudy  on  warming  and  separates  at  about  60°  C.  a  sticky  precipi- 
tate wliich  attaches  itself  to  the  sides  of  the  vessel  and  which  dissolves  at 
boiling  temperature  on  acidifying  the  urine;  the  precipitate  reappears  on 
cooling. 

In  close  relation  to  the  proteoses  stands  the  so-called  Bexce-Joxes 
proteid,  which  occurs  in  the  urine  in  rare  cases  in  disease  with  changes  in 
the  spinal  marrow.  It  gives  a  precipitate  on  heating  to  40-60°  C,  which  on 
further  heating  to  boiling  dissolves  again  more  or  less  completely,  depending 
upon  the  reaction  and  upon  the  amount  of  salt  present.  It  does  not  sepa- 
rate on  dialysis,  but  can  be  precipitated  from  the  urine  by  double  the 
volume  of  a  saturated  ammonium-sulphate  solution  or  by  alcohol.  It 
has  also  been  obtained  as  ciystals  (Grutterixk  and  de  Graaff,  ^Magx'US- 
Levy  1).  This  body  shows  a  somewhat  different  behavior  in  the  various 
cases  in  which  it  has  been  found  and  its  nature  has  not  been  explained. 
From  the  investigations  of  the  above-mentioned  and  other  experimenters 
(.MoiTESSiER,  Abderhaldex  and  Rostoski)  we  can  draw  the  conclusion 
that  this  proteid  is  similar  to  the  proteoses  in  several  reactions,  but  that 
nevertheless  it  stands  close  to  the  genuine  protein  bodies.  It  also  yields 
primar}'  as  well  as  secondaiy  proteoses  on  peptic  digestion  (Grutterixk 
and  DE  Graaff).  and  yields  the  same  hydrolytic  cleavage  products  as  the 
other  proteids  (Abderhaldex  and  Rostoski). 

Quantitative  Estimation  of  Proteid  in  Urine.  Of  all  the  methods  pro- 
posed thus  far,  the  coagulatiox  method  (boiling  with  the  addition  of 
acetic  acid)  when  performed  with  sufficient  care  gives  the  best  results. 
The  average  error  need  never  amount  to  more  than  0.01  per  cent,  and  it 
is  generally  smaller.  With  this  method  it  is  best  to  first  find  how  much 
acetic  acid  must  be  added  to  a  small  portion  of  the  urine,  which  has  l^een 
pre\iously  heated  on  the  water-bath,  to  completely  separate  the  proteid  so 
that  the  filtrate  will  not  respond  to  Heller's  test.  Then  coagulate 
20-50-100  c.c.  of  the  urine.  Pour  the  urine  into  a  beaker  and  heat  on 
the  water-bath,  add  the  required  quantity  of  acetic  acid  slowly,  stirring 
constantly,  and  heat  at  the  same  time.  Filter  while  warm,  wash  first 
with  water,  then  -^ath  alcohol  and  ether,  dr^^  and  weigh,  incinerate  and 
weigh  again.  In  exact  determinations  the  filtrate  must  not  give  Hel- 
ler's test. 

The  separate  estimation  of  globulixs  and  albumixs  is  done  by  care- 
fully neutralizing  the  urine  and  precipitating  -^nth  MgS04  added  to  satura- 
tion (Hammarstex)  .  or  simply  by  adding  an  equal  volume  of  a  saturated 


'  Magnus-Le\-y,  Zeitschr.  f.  physiol.  Chem..  30  (literature);  Grutteriiik  and  de 
Graaff,  ibid.,  34  and  46;  Moitessier,  Conipt.  rend.  soc.  biolog.,  o";  Abderhalden  and 
Rostoski,  Zeitschr.  f.  physiol.  Cliem.,  46. 


646  URINE. 

neutral  solution  of  ammonium  sulphate  (Hofmeister  andPoHLi).  The 
precipitate  consisting  of  globuhn  is  thoroughly  washed  with  a  saturated 
magnesium-sulphate  or  half-saturated  ammonium-sulphate  solution,  dried 
continuously  at  110°  C,  boiled  with  water,  extracted  with  alcohol  and 
ether,  then  dried,  weighed,  incinerated,  and  weighed  again.  The  quan- 
tity of  albumin  is  calculated  as  the  difference  between  the  quantity  ot 
globulin  and  the  total  proteids. 

Approximate  Estimation  of  Proteid  in  Urine.  Of  the  methods  sug- 
gested for  this  purpose  none  has  been  more  extensively  employed  than 
Esbach's. 

Esbach's^  Method.  The  acidified  urine  (with  acetic  acid)  is  poured 
into  a  specially  graduated  tube  to  a  certain  mark,  and  then  the  reagent 
(a  2  per  cent  citric-acid  and  1  per  cent  picric-acid  solution  in  water)  is  added 
to  a  second  mark,  the  tube  closed  with  a  rubber  stopper  and  carefully 
shaken,  avoiding  the  production  of  froth.  The  tube  is  allowed  to  stand 
twenty-four  hours,  and  then  the  height  of  the  precipitate  on  the  gradu- 
ation is  read  off.  The  reading  gives  directly  the  quantity  of  proteid  in 
1000  parts  of  the  urine.  Urines  rich  in  proteid  must  first  be  diluted  with 
water.  The  results  obtained  by  this  method  are,  however,  dependent 
■upon  the  temperature;  and  a  difference  in  temperature  of  5°  to  6.5°  C. 
may  cause  an  error  of  0.2-0.3  per  cent  deficiency  or  excess  in  urines  con- 
taining a  medium  quantity  of  proteid  (Christensen  and  Mygge^).  This 
method  is  only  to  be  used  in  a  room  in  which  the  temperature  may  be  kept 
nearly  constant.     The  directions  for  its  use  accompany  the  apparatus. 

Other  methods  for  the  approximate  estimation  of  proteid  are  the  optical 
methods  of  Christensen  and  Mygge,  of  Roberts  and  Stolnikow  as  modified 
by  Brandberg,  with  Heli,er's  test,  which  has  been  simplified  for  practical 
purposes  by  Mittelbach.  The  density  methods  of  Lang,  Huppert,  and  Zahor 
are  also  very  good.  In  regard  to  these  and  other  methods  we  refer  to  Huppert- 
Neubauer's  Harn-Analyse,  10.  Aufl. 

There  is  at  present  no  trustworthy  method  for  the  quantitative  estimation 
of  proteoses  and  peptone  in  the  urine. 

Nudeoalhumin  and  Mucin.  According  to  K.  Morner  traces  of  urinary 
mucoids  may  pass  into  solution  in  the  urine;  otherwise  normal  urine  con- 
tains no  mucin.  There  is  no  doubt  that  there  may  be  cases  where  true 
mucin  appears  in  the  urine;  in  most  cases  mucin  has  probably  been  mis- 
taken for  so-called  nucleoalbumin.  The  occurrence,  under  some  circum- 
stances, of  nucleoalbumin  in  the  urine  is  not  to  be  denied,  as  such  substances 
occur  in  the  renal  and  urinary  passages;  still  in  most  cases  this  nucleo- 
albumin, as  shown  by  K.  ^Morner,^  is  of  an  entirely  different  kind. 

Every  urine,  according  to  iMorner,  contains  a  little  proteid  and  in 
addition  substances  precipitating  proteid-     If  the  urine  freed  from  salts  by 

'  Hammarsten,  Pfliiger's  Arch.,  17;  Hofmeister  and  Pohl,  Arch,  f  exp.  Path.  u. 
Pharm.,  20. 

■  In  regard  to  the  literature  on  this  method  and  the  numerous  experiments  to  deter- 
mine its  value,  see  Huppert-Neubauer.  10  Aufl.,  853. 

'Christensen,  Virchow's  Arch.,  115. 

*  Skand.  Arch.  f.  Physiol.,  6. 


DETECTION    OF   NUCLEOALBUMIXS.  647 

dialysis  is  shaken  with  chloroform  after  the  addition  of  1-2  p.  m.  acetic 
acid,  a  precipitate  is  obtained  which  acts  Uke  a  nucleoalljiimin.  If  the 
acid  filtrate  is  treated  with  seralbumin,  a  new  and  similar  precipitate  is 
obtained  due  to  the  presence  of  a  residue  of  the  substance  ^\•hich  precipi- 
tates proteids.  The  most  important  of  these  proteid-precipitating  sub- 
stances is  chrondroitin-sulphuric  acid  and  nucleic  acid,  although  the  latter 
appears  to  a  much  smaller  extent.  TaurochoUc  acid  may  in  a  few  instances, 
especially  in  icteric  urines,  be  precipitated.  The  substances  isolated  by 
different  investigators  from  urine  by  the  addition  of  acet'c  acid  and  called 
''dissolved  mucin"  or  " nucleoalbumin"  are  considered  by  ]\1orxer  to  be 
a  combination  of  proteid  cliiefiy  witli  clirondroitin-sulphuric  acid,  and  to  a 
less  extent  with  nucleic  acid,  and  also  perhaps  with  taurocholic  acid. 

As  normal  urine  habitually  contains  an  excess  of  substances  capable  of 
precipitating  proteids,  it  is  apparent  that  an  increased  elimination  of  so- 
called  nucleoalbumin  may  be  caused  simply  by  an  augmented  excretion  of 
proteid.  Tliis  happens  to  a  still  greater  extent  in  cases  where  the  proteid 
as  well  as  the  proteid-precipitating  substance  is  eliminated  to  an  increased 
extent. 

Detection  of  so-called  Nucleoalbumins.  When  a  urine  becomes  cloudy 
or  precipitates  on  the  addition  of  acetic  acid,  and  when  it  gives  a  more 
typical  reaction  with  Heller's  test  after  the  dilution  of  the  urine  than 
before,  one  is  justified  in  making  tests  for  mucin  and  nucleoalbumin.  As 
the  salts  of  the  urine  interfere  considerably  with  the  precipitation  of  these 
substances  by  acetic  acid,  they  must  first  be  removed  by  dialysis.  As  large 
a  quantity  of  urine  as  possible  is  dialyzed  (with  the  addition  of  chloroform) 
until  the  salts  are  removed.  Then  acetic  acid  is  added  imtil  it  contains 
2  p.  m.,  and  the  mixture  allowed  to  stand.  The  precipitate  is  dissolved  in 
water  by  the  aid  of  the  smallest  possible  quantit}^  of  alkali  and  precipitated 
again.  In  testing  for  chrondroitin-sulphuric  acid  a  part  is  warmed  on  the 
w^ater-bath  with  about  5  per  cent  hydrochloric  acid.  If  positive  results  are 
obtained  on  testing  for  sulphuric  acid  and  a  reducing  substance,  then  chon- 
droproteid  was  present.  If  a  reducing  substance  can  be  detected  but  no 
sulphuric  acid,  then  mucin  is  probably  there.  If  it  does  not  contain  any 
sulphuric  acid  or  reducing  substance,  a  part  of  the  precipitate  is  exposed 
to  pepsin  digestion  and  another  part  used  for  the  determination  of  any 
■organic  phosphorus.  If  positive  results  are  obtained  from  these  tests, 
then  nucleoalbumin  and  nucleoproteid  must  be  differentiated  by  special 
tests  for  nuclein  bases.  No  positive  conclusion  can  be  drawn  except  by 
using  very  large  quantities  of  urine. 

NucleoMstonc.  In  a  case  of  pseudoleucaemia  A.  .Jolles  found  a  phosphorized 
protein  substance  which  he  considers  as  identical  with  nucleohistone.  Histone  is 
<>laimed  to  have  been  found  in  some  cases  by  Krehl  and  Matthes,  and  by  Kolisch 
and  BuRiAN.' 

'  JoUes,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30;  Krehl  and  Maithes,  Deutsch.  Arch. 
f.  kh'n.  Med,,  51;   Kolisch  and  Burian,  Zeitschr.  f.  klin.  Med.,  29. 


648  URINE. 

Blood  and  Blood-coloring  Matters.  The  urine  may  contain  blood  from 
hemorrhage  in  the  kidneys  or  other  parts  of  the  urinar>'  passages  (H^iL\- 
turia).  In  these  cases,  when  the  quantity  of  blood  is  not  ver>'  small,  the 
urine  is  more  or  less  cloudy  and  colored  reddish,  yellowish-red,  dirty  red, 
brownish  red,  or  dark  brown.  In  recent  hemorrhages,  in  which  the  blood 
has  not  decomposed,  the  color  is  nearer  blood-red.  Blood-corpuscles  may 
be  found  in  the  sediment,  sometimes  also  blood-casts  and  smaller  or  larger 
blood-clots. 

In  certain  cases  the  urine  contains  no  blood-corpuscles,  but  only  dis" 
solved  blood-coloring  matters,  hsemoglobin,  or,  and  indeed  quite  often, 
methaemoglobin  (h.emoglobixuria).  The  blood-pigments  appear  in  the 
urine  under  different  conditions,  as  in  dissolution  of  blood  in  poisoning  with 
arseniuretted  hydrogen,  chlorates,  etc.,  after  serious  burns,  after  trans- 
fusion of  blood,  and  also  in  the  periodic  appearance  of  hsemoglobinuria 
with  fever.  In  hsemoglobinuria  the  urine  may  also  have  an  abundant 
grayish-brown  sediment  rich  in  proteid  which  contains  the  remains  of  the 
stromata  of  the  red  blood-corpuscles.  In  animals  hsemoglobinuria  may 
be  produced  by  many  causes  which  force  free  hsemoglobin  into  the  plasma. 

To  detect  blood  in  the  urine,  we  make  use  of  the  microscope,  the  spec- 
troscope, the  guaiacum  test,  and  Heller's  or  Heller-Teichmann's  test. 

Microscopic  Investigation.  The  blood-corpuscles  may  remain  undis- 
solved for  a  long  time  in  acid  urine;  in  alkaline  urine,  on  the  contrarj',  they 
are  easily  changed  and  dissolved.  They  often  appear  entirely  unchanged  in 
the  sediment;  in  some  cases  they  are  distended  and  in  others  unequally 
pointed  or  jagged  like  a  thorn-apple.  In  hemorrhage  of  the  kidneys  a 
cylindrical  clot  is  sometimes  found  in  the  sediment  which  is  covered  with 
numerous  red  blood-corpuscles,  forming  casts  of  the  urinarj'  passages. 
These  formations  are  called  hlood-casts. 

The  spectroscopic  investigation  is  naturally  of  ver\^  great  value;  and  if 
it  be  necessary'  to  determine  not  only  the  presence  but  also  the  kind  of 
coloring-matter,  this  method  is  indispensable.  In  regard  to  the  optical 
behavior  of  the  various  blood-pigments  we  must  refer  to  Chapter  VI. 

Guaiacum  Test.  Mix  in  a  test-tube  equal  volumxes  of  tincture  of  guaia- 
cum and  old  turpentine  which  has  become  strongly  ozonized  by  the  action 
of  air  under  the  influence  of  light.  To  this  mixture,  which  must  not  have 
the  slightest  blue  color,  add  the  urine  to  be  tested.  In  the  presence  of  blood 
or  blood -pigments,  first  a  bluish-green  and  then  a  beautiful  blue  ring 
appears  where  the  two  liquids  meet.  On  shaking  the  mixture  it  becomes 
more  or  less  blue.  Normal  urine  or  one  containing  proteid  does  not  give 
this  reaction.  According  to  Liebermaxx  '  this  reaction  is  brought  about 
by  the  blood  pigments  acting  as  catalysators  upon  the  organic  peroxides 
existing  in  the  turpentine,  accelerating  the  decomposition  of  these  and  the 

•  Pfluger's  Arch.,  104. 


HiEMATOPORPHYRIN.  649 

active  oxygen  taken  up  by  the  guaiaconic  acid  which  is  oxidized  to  guaiac 
blue  (guaiaconic  acid  ozonide).  Urine  containing  pus,  even  when  no  blood 
is  present,  gives  a  blue  color  with  these  reagents ;  but  in  this  case  the  tincture 
of  guaiacum  alone,  without  turpentine,  is  colored  blue  by  the  urine  (Vitali  ^). 
This  is  at  least  true  for  a  tincture  that  has  been  exposed  for  some  time  to 
the  action  of  air  and  sunUght.  The  blue  color  produced  by  pus  differs 
from  that  produced  by  blood-coloring  matters  by  disappearing  on  heating 
the  urine  to  boiling.  A  urine  alkaUne  by  decomposition  must  first  be 
made  faintly  acid  before  performing  the  reaction.  The  turpentine  should 
be  kept  exposed  to  sunlight,  while  the  tincture  of  g-uaiacum  must  be  kept 
in  a  dark  glass  bottle.  These  reagents  to  be  of  use  must  be  controlled 
by  a  Uquid  containmg  blood.  With  positive  results,  however,  this  test  is 
not  absolutely  decisive,  tecause  other  bodies  may  give  a  similar  reaction; 
but  when  properly  performed  it  is  so  extremely  delicate  that  when  it  gives 
negative  results  any  other  test  for  blood  is  superfluous. 

Heller-Teichmann's  Test.  If  a  neutral  or  faintly  acid  urine  contain- 
ing blood  is  heated  to  boiling,  one  always  obtains  a  mottled  precipitate 
consisting  of  proteid  and  hiematin.  If  caustic  soda  is  added  to  the  boiling- 
hot  test,  the  liquid  l:)ecomes  clear  and  turns  green  when  examined  in  thin 
layers  (due  to  hsematin  alkali),  and  a  red  precipitate,  appearing  green  by 
reflected  light,  re-forms,  consisting  of  earthy  phosphates  and  haematin. 
This  reaction  is  called  Heller's  blood-test.  If  this  precipitate  is  col- 
lected after  a  time  on  a  small  filter,  it  may  he  used  for  the  hsemin  test  (see 
page  213).  If  the  precipitate  contains  only  a  little  blood-coloring  matter 
with  a  larger  quantity  of  earthy  phosphates,  then  wash  it  with  dilute  acetic 
acid,  which  dissolves  the  earthy  phosphates,  and  use  the  residue  for  the 
preparation  of  Teichmann's  hsemin  crystals.  If,  on  the  contra^v^  the 
amount  of  phosphates  is  very  small,  then  first  add  a  little  CaCl2  solution  tcv 
the  urine,  heat  to  boiling,  and  add  simultaneously  with  the  caustic  potash 
some  sodium-phosphate  solution.  In  the  presence  of  only  ver}'  small 
quantities  of  blood,  first  make  the  urine  very  faintly  alkaline  with  am- 
monia, add  tannic  acid,  acidify  with  acetic  acid,  and  use  this  precipitate  in 
the  preparation  of  the  hsemin  crv^stals  (Struve^). 

O.  and  R.  Adler  ^  have  recommended  leucomalachite  green  or  benzidine  in 
the  presence  of  peroxide  and  acetic  acid  as  especially  sensitive  reagents  for  blood. 
We  have  no  great  experience  thus  far  as  to  the  mode  of  use  of  these  reagents 
in  urine  investigations. 

Haematoporphyrin.  Since  the  occurrence  of  hsematoporphyrin  in  the 
urine  in  various  diseases  has  been  made  verv  probable  by  several  investi- 
gators, such  as  Neusser,  Stokvis,  ]\1acMunn,  Le  Nobel,  Copeman,  and 
others,'*  Salkowski  has  positively  shown  the  presence  of  this  pigment 
in  the  urine  after  sulphonal  intoxication.     It  was   first  isolated  in  a  pure 

*  See  Maly's  Jahresber.,  IS. 

^  Zeitschr.  f.  anal.  Chem.,  11. 

^  Zeitschr.  f.  phy.sioI.  Chem.,  41. 

^  A  very  complete  index  of  the  literature  on  hsematoporphyrin  in  the  urine  may  be 
found  in  R.  Zoja,  Su  qua'che  pigmento  di  alcune  urine,  etc.,  in  Arch.  Ital.  di.  clin. 
Med.,  189.3. 


650  URLXE. 

crystalline  state  by  Hammarsten  ^  from  the  urine  of  insane  women  after 
sulphonal  intoxication.  According  to  Garrod  and  Saillet^  traces  of 
haematoporphyrin  (Saillet's  urospectrin)  occur  regularly  in  normal 
urines.  It  is  also  found  in  the  urine  during  different  diseases,  although 
it  occurs  only  in  small  quantities.  It  has  been  found  in  considerable  quan- 
tities in  the  urine  after  the  lengthy  use  of  sulphonal. 

Urine  containing  haematoporphyrin  is  sometimes  only  slightly  colored, 
while  in  other  cases,  as  for  example,  after  the  use  of  sulphonal,  it  is  more  or 
less  deep  red.  In  these  last-mentioned  cases  the  color  depends,  in  greatest 
part,  not  upon  the  haematoporphyrin,  but  upon  other  red  or  reddish-brown 
pigments  which  have  not  been  sufficiently  studied. 

In  the  detection  of  small  quantities  of  haematoporphyrin  proceed  as 
suggested  by  Garrod.  Precipitate  the  urine  with  a  10  per  cent  caustic- 
soda  solution  (20  c.c.  for  every  100  c.c.  of  urine).  The  phosphate  precipi- 
tate containing  the  pigment  is  dissolved  in  alcohol-hydrochloric  acid  (15-20 
c.c.)  and  the  solution  investigated  by  the  spectroscope.  In  more  exact 
investigations  make  the  solution  alkaline  with  ammonia,  add  enough  acetic 
acid  to  dissolve  the  phosphate  precipitate,  shake  with  Ciiloroform,  which 
takes  up  the  pigment,  and  test  this  solution  with  the  spectroscope. 

In  the  presence  of  larger  quantities  of  haematoporphyrin  the  urine  is 
first  precipitated,  according  to  Salkowski,  with  an  alkaUne  barium- 
chloride  solution  (a  mixture  of  equal  volumes  of  barium-hydrate  solution, 
saturated  in  the  cold,  and  a  10  per  cent  barium-chloride  solution),  or,  accord- 
ing to  Hammarsten,^  with  a  barium-acetate  solution.  The  washed  pre- 
cipitate, which  contains  the  haematoporphyrin,  is  allowed  to  stand  some 
time  at  the  temperature  of  the  room  with  alcohol  containing  hydrochloric 
or  sulphuric  acid  and  then  filtered.  The  filtrate  shows  the  characteristic 
spectrum  of  haematoporphyrin  in  acid  solution  and  gives  the  spectrum  of 
alkaline  haematoporphyrin  after  saturation  with  ammonia.  If  the  alcoholic 
solution  is  mixed  with  chloroform  and  a  large  quantity  of  water  added  and 
carefully  shaken,  sometimes  a  lower  layer  of  chloroform  is  obtained  which 
contains  veiy  pure  haematoporphyrin,  while  the  upper  layer  of  alcohol  and 
water  contains  the  other  pigments  besides  some  haematoporphyrin. 

Other  methods  which  have  no  advantage  over  this  one  of  Garrod  have  been 
suggested  by  Riva  and  Zoja  as  well  as  Saillet.^ 

Baumstark  ^  found  in  a  case  of  leprosy  two  characteristic  coloring-matters 
in  the  urine,  "  urorubroha;matin  "  and  "  urofuscohsematin,"  which,  as  their  names 
indicate,  seem  to  stand  in  close  ralationship  to  the  blood-coloring  matters.  Uro- 
ruhrohcematin,  C68H94N8Fe202«,  contains  iron  and  shows  in  acid  solution  an  absorp- 
tion-band in  front  of  D  and  a  broader  one  back  of  D.     In   alkaline  solution  it 

^  Sa'kowski,  Zeitschr.  f .  physio  1.  Chem.,  lo;  Hammarsten,  Skand.  Arch,  f .  Physiol.,  3. 

-Garrod,  Joum.  of  Physio!.,  13  (contains  review  of  literature)  and  17;  Saillet, 
Revue  de  Medecine,  Ifi. 

^Salkowski,  1.  c;    Hammarsten,  1.  c. 

*  Riva  and  Zoja,  Maly's  Jahresber.,  24;  Saillet,  i.  c.  See  also  Nebeithau,  Zeitschr. 
f.  physiol.  Chem.,  27. 

'  Pfliiger's  Arch.,  9. 


PUS.  651 

shows  four  bands — behind  D,  at  E,  beyond  F,  and  behind  G.  It  is  not  soluble 
either  in  water,  alcohol,  ether,  or  cliloroform.  It  gives  a  beautiful  brownish-red 
non-dichroitic  liquid  with  alkalies.  Urofuscohcematin,  CsgHioeXgOon,  which  is  free 
from  iron,  shows  no  characteristic  spectrum;  it  dissolves  in  alkalies,  producing 
a  brown  color.  It  remains  to  be  proved  whether  these  two  pigments  are  related 
to  (impure)  haematoporphyrin. 

Melanin.  In  the  presence  of  melanotic  cancers  dark  pigments  are  some- 
times eliminated  with  the  urine.  K.  M()rxer  has  isolated  two  pigments  from 
such  a  urine,  of  which  one  was  soluble  in  warm  50-75  per  cent  acetic  acid,  while 
the  other,  on  the  contrary,  was  insoluble.  The  one  seemed  to  be  phymatorhusin 
(see  Chapter  XVI).  Usually  the  urine  does  not  contain  any  melanin,  but  a 
chromogen  of  melanin,  a  melanogen.  In  such  cases  the  urine  gives  Eislet's 
reaction,  becoming  dark-colored  with  oxidizing  agents,  such  as  concentrated 
nitric  acid,  potassium  bichromate,  and  sulphuric  acid,  as  well  as  with  free  sulphuric 
acid.  Urine  containing  melanin  or  melanogen  is  colored  black  by  a  ferric-chloride 
solution  (v.  Jaksch  ^). 

Urorosein,  so  named  by  Xenxki,^  is  a  urinary  coloring-matter  occurring  in 
various  diseases,  but  which  is  not  a  constituent  of  normal  urine.  The  pigment 
does  not  occur  preformed  in  the  urine,  but  first  makes  its  appearance  after  the 
addition  of  mineral  acids.  It  is  readily  soluble  in  water,  dilute  mineral  acids, 
ethyl  and  amyl  alcohol,  and  can  be  removed  from  the  acid  urine  by  shaking  with 
the  latter.  It  differs  from  indigo  red  in  the  following:  Alkalies  immediately 
decolorize  a  urorosein  solution,  but  not  an  indigo-red  solution.  Urorosein  is 
removed  from  its  amyl-alcohol  solution  by  shaking  with  dilute  alkali,  while  indigo 
red  is  not.  If  the  acid  urine  is  shaken  with  cliloroform,  indigo  red  is  taken  up, 
but  not  urorosein.  Urorosein  is  soon  decomposed  by  light  and  shows  a  sharply 
defined  absorption-band  between  D  and  E.  The  red  pigment  appearing  in  urines 
rich  in  skatol  after  the  addition  of  hydrochloric  acid  differs  from  urorosein  by 
being  insoluble  in  water,  but  readily  soluble  in  ether  and  chloroform.  The  state- 
ments in  regard  to  the  properties  of  skatol-red  are  somewhat  chvergent,  and  it  is 
therefore  difficult  to  state  a  positive  difference  between  urorosein  and  skatol-red. 

Pus  occurs  in  the  urine  in  various  inflammatory  affections,  especially 
in  catarrh  of  the  badder  and  in  inflammation  of  the  peh-is  of  the  kidneys 
or  of  the  urethra. 

Pus  is  best  detected  by  means  of  the  microscope.  The  pus-cells  are 
rather  easily  destroyed  in  alkaline  urines.  In  detecting  pus  we  make  use 
of  Donne's  pus  test,  which  is  performed  in  the  following  wa}"  Pour  off 
the  urine  from  the  sediment  as  carefully  as  possible,  place  a  small  piece  of 
caustic  alkali  on  the  sediment,  and  stir.  If  the  pus-cells  have  not  been 
previously  changed,  the  sediment  is  converted  by  tliis  means  into  a  sUmy 
tough  mass. 

The  pus-corpuscles  swell  up  in  alkaUne  urines,  dissolve,  or  at  least  are 
so  changed  that  they  cannot  be  recognized  under  the  microscope.  The 
urine  in  these  cases  is  more  or  less  shmy  or  fibrous,  and  the  proteid  can  be 
precipitated  in  large  flakes  by  acetic  acid,  so  that  it  might  possibly  be  mis- 
taken for  mucin.  The  closer  investigation  of  the  precipitate  produced  by 
acetic  acid,  and  especially  the  appearance  or  non-appearance  of  a  reducing 
substance  after  boihng  it  with  a  mineral  acid,  demonstrates  the  nature  of 
the  precipitated  substance.      Urine  containing  pus  always  contains  proteid. 

'  K   Murner.  Zeitschr.  f.  physiol.  Chem..  11;    v.  Jaksch.  ibid.,  13. 
-  Nencki  and  Sieber,  Joum.  f.  prakt.  Chem.  (X.  F.),  2G. 


652  URINE. 

Bile-acids.  The  reports  in  regard  to  the  occurrence  of  bile-acids  in  the 
urine  under  physiological  conditions  do  not  agree.  According  to  Dragen- 
DORFF  and  Hone  traces  of  bile-acids  occur  in  the  urine ;  according  to  Mackay 
and  V.  Udranszky  and  K.  ]\Iorner  ^  they  do  not.  Pathologically  they 
are  present  in  the  urine  in  hepatogenic  icterus,  although  not  invariably. 

Detection  of  Bile-acids  in  the  Urine.  Pettenkofer's  test  gives  the 
most  decisive  reaction;  but  as  it  gives  similar  color  reactions  with  other 
bodies,  it  must  be  supplemented  by  the  spectroscopic  investigation.  The 
direct  test  for  bile-acids  is  easily  performed  after  the  addition  of  traces  of 
bile  to  a  normal  urine.  But  the  direct  detection  in  a  colored  icteric  urine 
is  more  difficult  and  gives  very  misleading  results;  the  bile-acid  must  there- 
fore always  be  isolated  from  the  urine.  This  may  be  done  by  the  following 
method  of  Hoppe-Seyler,  which  is  slightly  modified  in  non-essential 
points. 

Hoppe-Seyler's  ^Method.  Concentrate  the  urine  and  extract  the 
residue  with  strong  alcohol.  The  filtrate  is  freed  from  alcohol  by  evapo- 
ration and  then  precipitated  by  basic  lead  acetate  and  ammonia.  The 
washed  precipitate  is  treated  with  boiling  alcohol,  filtered  hot,  the  filtrate 
treated  with  a  few  drops  of  soda  solution,  and  evaporated  to  dryness.  The 
dry  residue  is  extracted  with  absolute  alcohol,  filtered,  and  an  excess  of 
ether  added.  The  amorphous  or,  after  a  longer  time,  crystalline  precipi- 
tate consisting  of  the  alkali  salts  of  the  biUarj'^  acids  is  used  in  performing 
Pettenkofer's  test. 

Haycraft  has  suggested  a  reaction  for  clinical  jDurposes  which  consists  in 
sprinkling  flowers  of  sulphur  upon  the  urine.  In  icteric  urine  the  powder  quickly 
sinks  to  the  bottom,  while  in  normal  urine  it  remains  on  the  surface.  The  vp,lue 
of  this  test  is  stUl  questioned. 

Bile-pigments  occur  in  the  urine  in  different  forms  of  icterus.  A 
urine  containing  bile-pigments  is  always  abnormally  colored — ^yellow, 
yellowish  brown,  deep  brown,  greenish  yellow,  greenish  brown,  or  nearly 
pure  green.  On  shaking  it  froths  and  the  bubbles  are  yellow  or  yellowish 
green  in  color.  As  a  rule  icteric  urine  is  somewhat  cloudy,  and  the  sedi- 
ment is  frequently,  especially  when  it  contains  epithelium-cells,  rather 
strongly  colored  by  the  bile-pigments.  In  regard  to  the  occurrence  of 
urobihn  in  icteric  urine  see  p.  604. 

Detection  of  Bile-coloring  Matters  in  Urine.  ^lany  tests  have  been  pro- 
posed for  the  detection  of  these  substances.  Ordinarily  we  obtain  the  best 
results  either  with  Gmelin's  or  wath  Huppert's  test. 

Gmelin's  test  may  be  applied  directly  to  the  urine;  but  it  is  better  to 
use  Rosenbach's  modification.  Filter  the  urine  through  a  very  small  filter, 
which  becomes  deeply  colored  from  the  retained  epithelium-cells  and  bodies 
of  that  nature.  After  the  Uquid  has  entirely  passed  through  apply  to  the 
inside  of  the  filter  a  drop  of  nitric  acid  which  contains  only  very  little 
nitrous  acid.     A  pale-yellow  spot  will  he  formed  wiiich  is  surrounded  by 

'Cited  from  Huppert-Neubauer,  Ham- Analyse,  10.  Aufl.   229. 


BILE-PIG  MEXTS.  653 

colored  rings  which  appear  yellowish  reel,,  ^'iolet,  blue,  and  green  from 
within  outward.  Tliis  modification  is  veiy  deUcate,  and  it  is  hardly  possi- 
ble to  mistake  indican  and  other  coloring-matters  for  the  bile-pigments. 
Several  other  modifications  of  Gmelin's  direct  test,  e.  g.,  with  concentrated 
sulphuric  acid  and  nitrate,  etc.,  have  been  proposed,  but  they  are  neither 
simpler  nor  more  dehcate  than  Rosexbach's  modification. 

Huppert's  Reaction.  In  a  dark-colored  urine  or  one  rich  in  indican 
good  results  are  not  always  obtained  with  Gmelix's  test.  In  such  cases, 
as  also  in  urines  containing  blood-coloring  matters  at  the  same  time,  the 
urine  is  treated  with  lime-water,  or  first  with  some  CaCU  solution,  and  then 
with  a  solution  of  soda  or  ammonium  carbonate.  The  precipitate  which 
contains  the  bile-coloring  matters  is  filtered,  washed,  dissolved  in  alcohol 
which  contains  5  c.c.  of  concentrated  hydrochloric  acid  in  100  c.c.  (I.  Muxk), 
and  heated  to  boiUng  when  the  solution  becomes  green  or  bluish  green. 
According  to  Xakayama  ^  this  reaction  is  more  deUcate  on  using  a  mixture 
of  ferric  chloride,  acid,  and  alcohol. 

Hammarstex's  React ioji.  For  ordinaiy  cases  it  is  sufficient  to  add  a 
few  drops  of  urine  to  about  2-3  c.c.  of  the  reagent  (see  page  322).  when  the 
mixture  immediately  after  shaking  turns  a  beautiful  green  or  bluish  green, 
which  color  remains  for  several  days.  In  th€  presence  of  only  vers*  small 
quantities  of  bile-pigments,  especially  when  blood  or  other  pigments  are 
simultaneously  present,  pour  about  10  c.c.  of  the  acid  or  nearly  neutral 
(not  alkaline)  urine  into  the  tube  of  a  small  centrifugal  machine  and  add 
BaClo  solution  and  centrifuge  for  about  one  minute.  The  liquid  is  decanted 
off  and  the  sediment  stirred  with  about  1  c.c.  of  the  reagent  and  centri- 
fuged  again.  A  beautiful  green  solution  is  obtained,  which  may  be  changed 
by  the  addition  of  increased  quantities  of  the  acid  mixture  to  blue,  ^'iolet. 
red,  and  reddish  yellow.  The  gree^i  color  may  be  obtained  in  the  presence 
of  1  part  bile-pigment  in  500,000-1,000,000  parts  urine.  In  the  presence 
of  large  amounts  of  other  pigments  calcium  chloride  is  better  suited  than 
barium  chloride. 

Bouma2  has  suggested  the  use  of  alcohol  containing  ferric  chloride 
and  hydrochloric  acid  instead  of  the  above-mentioned  acid  mixture.  He 
has  also  worked  out  a  colorimetric  method  of  quantitative  estimation  of 
bilinibin  in  urine  by  means  of  this  reagent. 

The  veiy  delicate  reaction  suggested  by  Jolles  is  unfortunately  not 
ser^'iceable  on  account  of  the  formation  of  froth,  especially  in  the  presence 
of  proteid  and  blood-pig-nients ;  but  he  has  changed  it  by  centrifuging  the 
urine  with  chloroform  and  barium  chloride  and  suspending  the  chloroform- 
barium  residue  in  alcohol;  after  which  he  treats  it  with  a  solution  of  iodine 
and  mercuric  chloride  in  alcohol  containing  hydrochloric  acid.^  The  color 
becomes  green  or  bluish  green.     This  test  seems  to  be  good. 

Stokvis's  reaction  is  especially  valuable  as  a  control  test  in  those  cases 
in  which  the  urine  contains  only  xevy  Uttle  bile-coloring  matter  together 
with  larger  quantities  of  other  coloring-matters.  The  test  is  performed  as 
follows:    20-30  c.c.  of  urine  is  treated  with  5-10  c.c.  of  a  solution  of  zinc 

*  Munk,  Arch.  f.  (.Ajiat.  u.)  Physiol.,  1S9S;  Xakayama,  Zeitschr.  f.  physiol. 
Chem.,  36 

2  Deutsch.  med.  Wochenschr.,  1902  and  1904. 
'  Deutsch.  Arch,  f .  k'in.  Med.,  78. 


654  URINE. 

acetate  (1:5).  The  precipitate  is  washed  on  a  small  filter  with  water  and 
then  dissolved  in  a  little  ammonia.  The  new  filtrate  gives,  either  directly 
or  after  it  has  stood  a  short  time  in  the  air  until  it  has  a  peculiar  brownish- 
green  color,  the  absorption-bands  of  bilicyanin  (see  page  322).  This  reac- 
tion is  unfortunately  not  sufficiently  delicate. 

^lany  other  reactions  for  bile-coloring  matters  in  the  urine  have  been 
proposed;  but  as  those  above  mentioned  are  sufficient,  it  is  perhaps  only 
necessary  to  give  here  a  few  of  the  other  reactions  without  entering  into 
details. 

Smith's  Reaction.  Pour  carefully  over  the  uiine  some  tincture  of  iodine, 
whereby  a  green  ring  appears  between  the  two  liquids.  The  urine  may  also  be 
shaken  with  the  tincture  of  iodine  until  it  has  a  green  color. 

Ehrlich's  Test.  First  mix  the  urine  with  an  equal  volume  of  dilute  acetic 
acid  and  then  add  drop  by  drop  a  solution  of  sulphodiazobenzene.  The  acid 
mixture  becomes  dark  red  in  the  presence  of  bilirubin,  and  this  color  becomes 
bluish  \'iolet  on  the  addition  of  glacial  acetic  acid.  The  sulphodiazobenzene  is 
prepared  by  mixing  1  gram  of  sulphanilic  acid,  15  c.c.  of  hydrochloric  acid,  and 
0.1  gram  of  sodium  nitrite ;  this  solution  is  diluted  to  1  liter  with  water.  This 
test  is  not  successful  and  positive  when  directly  applied,  if  the  urine  is  rich  in  other 
pigments. 

Medicinal  coloring-matters  produced  from  santonin,  rhubarb,  senna,  etc., 
may  give  an  abnormal  color  to  the  urine  and  may  be  mistaken  for  bile-pigments, 
or,  in  alkaline  urines,  perhaps  for  blood-coloring  matters.  If  hydrochloric  acid 
is  added  to  such  a  urine,  it  becomes  yellow  or  pale  yellow,  while  on  the  addi- 
tion of  an  excess  of  alkali  it  takes  on  a  more  or  less  beautiful  red  color. 


Sugar  in  Urine. 

The  occurrence  of  traces  of  dextrose  in  the  urine  of  perfectly  healthy 
persons  has  been,  as  above  stated  (page  608),  quite  positively  proved.  If 
sugar  appears  in  the  urine  in  constant  and  especially  in  large  quantities,  it 
must  be  considered  as  an  abnormal  constituent.  In  a  previous  chapter 
several  of  the  principal  causes  of  glycosuria  in  man  and  animals  were  men- 
tioned, and  the  reader  is  referred  to  Chapters  VIII  and  IX  for  the  essential 
facts  in  regard  to  the  appearance  of  sugar  in  the  urine. 

In  man  the  appearance'of  dextrose  in  the  urine  has  been  observed  under 
various  pathological  conditions,  such  as  lesions  of  the  brain  and  especially 
of  the  medulla  oblongata,  abnormal  circulation  in  the  abdomen,  diseases 
of  the  heart,  lungs  and  liver,  cholera,  and  many  other  diseases.  The 
continued  presence  of  sugar  in  human  urine,  sometimes  in  very  considerable 
quantities,  occurs  in  diabetes  mellitus.  In  this  disease  there  may  be 
an  elimination  of  1  kilogram  or  even  more  of  dextrose  per  day.  In  the 
beginning  of  the  disease,  when  the  quantity  of  sugar  is  still  very  small, 
the  urine  often  does  not  ajjpear  abnormal.  In  the  more  developed,  typical 
cases  the  quantity  of  urine  voided  increases  considerably,  to  3-6-10  liters 
per  day.  The  percentage  of  the  physiological  constituents  is  as  a  nde  very 
low,  while  their  al^solute  daily  quantity  is  increased.     The  urine  is  pale. 


SUGAR  LN    URIXE.  655 

but  of  a  high  specific  gravity,  1.030-1.040  or  even  higher.  The  high  spe- 
cific gravity  depends  upon  the  quantity  of  sugar  present,  which  varies  in 
different  cases,  but  may  reach  10  per  cent.  The  urine  is  therefore  charac- 
terized in  typical  cases  of  diabetes  by  the  very  large  quantity  voided,  by 
the  pale  color  and  high  specific  gravity,  and  by  its  containing  sugar. 

That  the  urine  after  the  introduction  into  the  system  of  certain  medic- 
inal agents  or  poisonous  bodies  contains  reducing  substances,  conjugated 
glucuronic  acids,  which  may  be  mistaken  for  sugar,  has  already  been  men- 
tioned. 

The  properties  and  reactions  of  dextrose  have  been  considered  in  a 
previous  chapter,  and  it  remains  but  to  mention  the  methods  for  the  detec- 
tion and  quantitative  determination  of  dextrose  in  the  urine. 

The  detection  oj  stigar  in  the  urine  is  ordinarily,  in  the  presence  of  not 
too  small  quantities,  a  very  simple  task.  The  presence  of  only  ver}-  small 
quantities  may  make  its  detection  sometimes  ver\'  difficult  and  laborious. 
A  urine  containing  proteid  must  first  have  the  proteid  removed  by  coagu- 
lation with  acetic  acid  and  heat  before  it  can  be  tested  for  sugar. 

The  tests  which  are  most  frequentl}'  emplo^-ed  and  are  especially  recom- 
mended are  as  follows: 

Tromm]:r's  Test.  In  a  typical  diabetic  urine  or  one  rich  in  sugar  this 
test  succeeds  well,  and  it  may  be  performed  in  the  manner  suggested  on 
page  116.  This  test  may  lead  to  very  great  mistakes  in  urines  poor  in  sugar, 
especially  wiien  they  have  at  the  same  time  normal  or  increased  amounts  of 
physiological  constituents,  and  therefore  it  cannot  be  recommended  to 
physicians  or  to  persons  inexperienced  in  such  work.  Normal  urine  con- 
tains reducing  substances,  such  as  uric  acid,  creatinine,  and  others,  and 
therefore  a  reduction  takes  place  in  all  urines  on  using  this  test.  A  separa- 
tion of  copper  suboxide  does  not  generally  occur,  but  still  if  one  varies  the 
proportion  of  the  alkali  to  the  copper  sulphate  and  boils,  there  takes  place 
an  actual  separation  of  sulfoxide  in  normal  urines,  or  a  j^eculiar  yellowish 
red  liquid  due  to  finely  di\-ided  cuprous  hydrate.  This  occurs  especially 
on  the  addition  of  much  alkali  or  too  much  copper  sulphate,  and  by  careless 
manipulation  the  inexperienced  worker  may  therefore  sometimes  obtain 
apparently  positi\e  results  in  a  normal  urine.  On  the  other  hand,  as  the 
urine  contains  substances,  such  as  creatinine  and  ammonia  (from  the  urea)^ 
which  in  the  presence  of  only  a  little  sugar  may  keep  the  cojjper  suboxide 
in  solution,  the  investigator  may  easily  overlook  small  quantities  of  sugar 
that  may  he  present. 

Trommer's  test  may  of  course  be  made  positive  and  useful,  even  in  the 
presence  of  very  small  amounts  of  sugar.  l)y  using  the  modification  su/:- 
gested  by  Worm  Duller.  As  this  modification  is  rather  compUcatcd 
and  requires  much  practice  and  exactness,  it  is  probably  rarely  employed 
by  the  busy  physician.     The  following  test  is  to  be  preferred. 

Almi&x's  bismuth  test,  which  recently  has  been  incorrectly  called  Nylax- 
DEr's  test,  is  performed  with  the  alkaline-bismuth  solution  prepared  as 
above  described  (page  116).  For  each  test  10  c.c.  of  urine  is  taken  and 
treated  with  1  c.c.  of  the  ])ismuth  solution  and  boiled  for  a  few  minutes. 
In   the  presence  of  sugar  the  urine    l^ecomes  dark    vcllow    or    vellowish 


656  URIiXE. 

brown.  Then  it  grows  darker,  cloudy,  dark  brown,  or  nearly  black,  and 
non-transparent.  After  a  longer  or  shorter  time  a  black  deposit  appears, 
the  supernatant  liquid  gradually  clears,  but  still  remains  colored.  In  the 
presence  of  only  veiy  little  sugar  the  test  does  not  become  black  or  dark 
brown,  but  simply  deej^er-colored,  and  not  until  after  some  time  is  there 
seen  on  the  upper  layer  of  the  phosphate  precipitate  a  dark  or  black  layer 
(of  bismuth?).  In  the  presence  of  much  sugar  a  larger  amount  of  the 
reagent  may  l^e  used  without  disadvantage.  In  a  urine  poor  in  sugar  only 
1  c.c.  of  the  reagent  for  every  10  c.c.  of  the  urine  must  be  employed. 

Small  amounts  of  proteid  may  retard  this  reaction  and  reduce  the  deli- 
cacy of  the  test.  Large  quantities  of  proteid  may,  however,  give  rise  to  an 
error  b}^  forming  bismuth  sulphide,  and  therefore  it  must  always  be  first 
removed.  The  statement  of  Bechhold  that  mercury  compounds  in  the  urine 
disturb  the  test  has  not  been  substantiated  by  Zeidijtz  i  on  properly  })er- 
forming  the  test.  Those  sources  of  error  which  in  Trommer's  test  are 
caused  by  the  presence  of  uric  acid  and  creatinine  are  removed  by  using 
this  test.  The  bismuth  test  is,  moreover,  readily  performed,  and  on  this 
account  is  to  be  recommended  to  the  physician. 

The  bumping  and  ejection  of  the  fluid  can  be  readily  prevented  by  heating 
over  a  very  small  flame  after  the  test  has  been  brought  to  a  boil  and  by  gently 
shaking  the  contents  of  the  not  too  narrow  test-tube.  The  recommendation 
of  heating  for  a  longer  time  in  the  water-bath,  fifteen  minutes  or  more,  is  to  be 
discarded,  as  the  delicacy  of  the  test  is  thereby  so  much  increased  that  it  gives  a 
reaction  with  a  physiological  sugar  content  of  0.02  per  cent. 

When  the  amount  of  sugar  in  the  urine  is  not  less  than  0.1  per  cent  a 
positive  reaction  is  obtained  if  the  test  is  boiled  for  2-3  minutes  and  then 
allowed  to  stand  quietly  for  5  minutes.  The  phosphate  precipitate  is  then 
black  or  nearly  Ijlack.  In  detecting  smaller  quantities  of  sugar— O.05 
per  cent,  the  test  as  a  rule  must  be  boiled  longer — about  5  minutes. 

The  value  of  this  test  hes  in  the  fact  that  it  positively  detects  small 
quantities  of  sugar — 0.1  per  cent  or  somewhat  less.  Equally  with 
Trommer's  test  it  is  a  reduction  test,  and  shows  also  certain  other  reducing 
bodies  besides  the  sugar.  These  bodies  are  certain  conjugated  glucuronic 
acids  which  may  appear  in  the  urine.  After  the  use  of  certain  therapeutic 
agents,  such  as  rhubarb,  senna,  antipyrine,  salol,  turpentine  and  others, 
the  bismuth  test  gives  positive  results.  From  this  it  follows  that  we  should 
never  be  satisfiecl  with  this  test  alone,  especially  when  the  reduction  is  not 
very  great.  When  this  test  gives  negative  results  the  urine  can  be  con- 
sidered from  a  clinical  standpoint  as  free  from  sugar,  and  when  it  gives 
positive  results  other  tests  must  be  applied.  Among  these  the  fermentation 
test  and  the  polarization  test  are  of  special  value. 

The  question  in  what  degree  the  use  of  therapeutic  agents  affects  the  Worm- 
Ml'iLLER  test  has  been  only  slightly  investigated.  That  normal  urines  some- 
times give  the  bismuth  test  is  a  common  experience,  and  these  urines  according 
to  the  experience  of  Hammarsten  and  certain  other  observers,  as  a  rule  also 
give  the  WoRM-MtJLLER  test  when  properly  performed.  There  does  not  seem  to 
be  any  doubt  that  in  many  of  these  cases  it  is  due  to  an  increased  quantity 
of  physiological  sugar  in  the  urine.  Further  investigations  on  this  point  are 
very  desirable. 

'Bechhold,  Zeitschr.  f.  physiol.  Cliem.,  46;   Zeidlitz,  unpublished  investigations. 


SUGAR  IN   URINE.  657 

Fermentation  Test.  On  using  this  test  the  process  must  vaty  aci-ord- 
ing  as  the  bismuth  test  shows  small  or  large  quantities  of  sugar.  If  a  rather 
strong  reduction  is  obtained,  the  urine  may  be  treated  with  yeast  and  the 
presence  of  sugar  determined  b}^  the  generation  of  carbon  dioxide.  In 
this  case  the  acid  urine,  or  that  faintly  acidified  with  a  little  sulphuric 
or  hydrochloric  acid,  is  treated  with  compressed  yeast  or  yeast  which  has 
previously  been  washed  by  decantation  with  water.  Pour  this  urine  to 
Avhich  the  yeast  has  been  added  into  a  Schrotter's  gas-burette  or  a  Lohn- 
STEix's  saccharimeter  (see  below).  As  the  fermentation  proceeds,  the 
carbon  dioxide  collects  in  the  upper  part  ot  the  tube,  while  a  corresponding 
quantity  of  liquid  is  expelled  below.  As  a  control  in  this  case  two  similar 
tests  must  be  made,  one  with  normal  urine  and  j'east  to  learn  the  quantity 
of  gas  usually  developed,  and  the  other  with  a  sugar  solution  and  yeast 
to  determine  the  activity  of  the  yeast. 

If,  on  the  contrary-,  only  a  faint  reduction  with  the  bismuth  test  is 
found,  no  positive  conclusion  can  be  drawn  from  the  absence  of  any  carbon 
dioxide  or  the  appearance  of  a  ver\'  insignificant  quantity.  The  urine 
absorbs  considerable  amounts  of  carbon  dioxide,  and  in  the  presence  of 
only  small  amounts  of  sugar  the  fermentation  test  as  above  performed 
may  lead  to  negative  or  inaccurate  results.  In  this  case  proceed  in  the 
folloT\-ing  wa}':  Treat  the  acid  urine,  or  urine  which  has  been  faintly 
-acidified  -uith  a  little  sulphuric  acid,  with  yeast  whose  acti^^ty  has  been 
tested  by  a  special  test  on  a  sugar  solution,  and  allow  it  to  stand  24-30  hours 
iit  about  30°.  Then  test  again  with  the  bismuth  test,  and  if  the  reaction 
noAv  gives  negative  results,  then  sugar  was  previously  present.  But  if  the 
reaction  continues  to  give  positive  results,  then  it  shows,  if  the  yeast  is 
active,  the  presence  of  other  reducing,  unfermentable  substances. 

In  performing  the  fermentation  test  care  should  be  taken  that  the  urine 
be  acid  before  as  well  as  after  fermentation.  If  the  reaction  becomes  alka- 
line during  fermentation  (alkaline  fermentation),  then  the  test  must  be 
discarded.  The  vessel  must  be  perfectly  clean  and  strongly  heated  before 
use.     To  make  sure  the  urine  may  be  boiled  before  fermentation.^ 

If  a  good  polariscope  is  at  hand  it  must  not  be  forgotten  to  control  the 
results  of  the  fermentation  bv  determining  the  rotation  before  and  after 
fermentation.  The  phenylhydrazine  test  also,  in  many  othen\-ise  doubtful 
cases,  gives  good  service  in  testing  urines  for  sugar. 

Phenylhydrazine  Test.  According  to  v.  Jaksch  this  test  is  performed 
in  the  following  way:  Add  in  a  test-tube  containing  6-8  c.c.  of  the  urine 
two  knife-points  of  phenylhydrazine  hydrochloride  and  three  knife-points 
of  sodium  acetate,  and  when  the  salts  do  not  dissolve  on  warming  add 
more  water.  The  test-tube  is  placed  in  boiling  water  and  warmed  on  the 
Avater-bath.  It  is  then  placed  in  a  beaker  of  cold  water.  If  the  quantity 
of  sugar  present  is  not  too  small,  a  yellow  cr}-stalline  precipitate  is  now 
obtained.  If  the  precipitate  appears  amorphous,  there  are  found,  on 
looking  at  it  under  the  microscope,  yellow  needles  singly  and  in  groups. 
If  very  little  sugar  is  present,  pour  the  test  into  a  conical  glass  and  examine 
the  sediment.     In  this  case  at  least  a  few  phenylglucosazone  cr\-stals  are 

'  On  the  perfonnance  of  the  fermentution  test  and  certain  sources  of  error,  see 
Salkowski,  BerHu.  klin.  Wochenschr.,  1905  (Ewald-Festnummer),  and  Pfliiger,  Pfltiger's 
Arch,  105. 


658  URINE. 

found,  while  the  occurrence  of  larger  and  smaller  yellow  plates  or  highly 
refractive  brown  globules  does  not  show  the  presence  of  sugar.  This 
reaction  is  very  reliable,  and  by  it  the  presence  of  0.03  per  cent  sugar  can 
be  detected  (Rosenfeld,  Geyer  i).  In  doubtful  cases  where  certainty 
is  desired,  prepare  tlie  crystals  from  a  large  quantity  of  urine,  dissolve  them 
on  the  filter  by  pouring  over  them  hot  alcohol,  treat  the  filtrate  with  water, 
and  boil  off  the  alcohol.  Still  better,  the  precipitate  is  dissolved,  according 
to  Neuberg,  in  some  pyridine,  and  again  precipitated  as  crystals  by  the 
addition  of  benzene,  ligroin,  or  ether.  If  the  characteristic  yellow  crystal- 
line needles,  whose  melting-point  (204-205°  C.)  may  also  be  determined,  are 
now  obtained,  then  this  test  is  decisive  for  the  presence  of  sugar.  It  must 
not  be  forgotten  that  levulose  gives  the  same  osazone  as  dextrose,  and 
that  a  further  investigation  is  necessary  in  certain  cases.. 

The  following  modification  by  A.  Neumann  2  is  simple,  practical,  and 
at  the  same  time  sufficiently  delicate.  5  c.c.  of  the  urine  is  treated  with 
2  c.c.  of  acetic  acid  (30  per  cent)  saturated  with  sodium  acetate,  2  drops 
of  pure  phenylhydrazine  added,  and  the  mixture  boiled  in  a  test-tube  until 
it  measures  3  c.c.  After  quickly  cooling  warm  again  and  then  allow  it  to 
cool  slowly.  After  5-10  minutes  beautifulh^  formed  crystals  are  obtained 
even  in  the  presence  of  only  0.02  per  cent  sugar.  According  to  the  exjjeri- 
ence  of  Hammarsten  tliis  modification,  even  in  the  presence  of  0.1  per  cent 
sugar  in  concentrated  urines,  does  not  always  give  a  positive  reaction. 

The  value  of  the  phenylliydrazine  test  has  l^een  considerably  debated, 
and  the  objection  has  been  made  that  glucuronic  acids  also  give  a  similar 
precipitate.  A  confounding  with  glucuronic  acid  is,  according  to  Hirschl, 
not  to  be  apprehended  when  the  test  is  not  heated  in  the  water-bath  for  too 
short  a  time  (one  hour).  Kistermann  found  this  precaution  insufficient, 
and  Roos  states  that  the  phenylhydrazine  test  always  gives  a  positive 
result  with  human  urine,  which  coincides  with  E.  Holmgren's  ^  and  Ham- 
marsten's  experience.  This  test  only  shows  a  non-physiological  quan- 
tity of  sugar  when  a  rather  abundant  crystallization  is  obtained  from  a 
small  quantity  of  urine  (about  5  c.c).  Too  great  a  delicacy  of  test  is  not 
to  be  recommended. 

Rubner's  test  is  performed  as  follows:  The  virine  is  precipitated  by 
an  excess  of  a  concentrated  lead-acetate  solution  and  the  filtrate  carefully 
treated  with  enough  ammonia  to  produce  a  flocculent  precipitate.  It  is 
then  heated  to  boiling,  when  the  precipitate  becomes  flesh-colored  or  pink 
in  the  presence  of  sugar. 

Polarization.  This  test  is  of  great  value,  especially  as  in  many  cases  it 
quickly  differentiates  between  dextrose  and  other  reducing,  sometimes 
levogyrate,  su]:)stances,  such  as  the  conjugated  glucuronic  acids.  In  the 
presence  of  only  very  little  sugar  the  value  of  this  test  depends  on  the  deli- 
cacy of  the  instrument  and  the  dexterity  of  the  observer.  As  a  urine  which 
shows  no  rotation  or  is  actually  faintly  levorotator\^  may  contain  0.2  per 

'  Rosenfeld,  Deutsch.  ir.ed.  Wochenschr.,  1888;  Geyer,  cited  from  Roos,  Zeitschr. 
f.  physiol.  Chem.,  15. 

•  Arcli.  f.  (Anat.  u.)  Plij^siol.,  1899,  Suppl.  See  also  Margulies,  Berlin,  klin.  Woclien- 
schr.,  1900. 

^  Hirschl,  Zeitschr.  f.  pliysiol.  Clieni.,  14;  Kistermann,  Deutsch.  Arch:  f.  klin. 
Med.,  oO;   Roos,  1.  c;   Holmgren,  Maly's  Jah;esber.,  27. 


SUGAR  IN    URINE.  659 

cent  sugar  or  perhaps  even  more,  this  test  must  be  combined  with  the  fer- 
mentation test  if  we  are  seeking  ver}-  small  amounts  of  sugar.  The  sugar 
in  these  cases  can  be  detected  only  by  the  use  of  a  very  accurate  and  deli- 
cate instrment.  Tliis  method  is  in  man}'  cases  not  serviceable  for  the 
physician. 

If  small  quantities  of  sugar  are  to  be  isolated  from  the  urine,  precipitate 
the  urine  first  with  sugar  of  lead,  filter,  precipitate  the  filtrate  with  am- 
moniacal  basic  lead  acetate,  wash  this  precipitate  with  water,  decompose  it 
with  H2S  when  suspended  in  water,  concentrate  the  filtrate,  treat  it  with 
strong  alcohol  until  it  is  SO  vol.  per  cent,  filter  when  necessary-,  and  add 
an  alcoholic  caustic-alkaU  solution.  Dissolve  the  precipitate  consisting  of 
saccharates  in  a  little  water,  precipitate  the  potash  by  an  excess  of  tartaric 
acid,  neutralize  the  filtrate  with  calcium  carbonate  in  the  cold,  and  filter. 
The  filtrate  may  be  used  for  testing  with  the  polariscope  as  well  as  for  the 
fermentation,  bismuth,  and  phenylhydrazine  tests.  The  presence  of  dex- 
trose may  be  detected  by  this  same  process  in  animal  fluids  or  tissues  from 
which  the  proteids  have  been  removed  by  coagxilation  or  by  the  addition 
of  alcohol. 

In  the  isolation  of  sugar  and  carbohydrates  from  the  luine  the  benzoic- 
acid  esters  of  the  same  may  be  prepared  according  to  Baumaxx's  method. 
The  urine  is  made  alkaline  ^^ith  caustic  soda  to  precipitate  the  earthy  phos- 
phates, the  filtrate  treated  with  10  c.c.  of  benzoyl  chloride  and  120  c.c. 
of  10  I3er  cent  caustic-soda  solution  for  ever}-  100  c.c.  of  the  filtrate  (Reix- 
BOLD^),  and  shaken  vuitil  the  odor  of  benzoyl  chloride  has  disappeared. 
After  standing  sufficiently  long  the  ester  is  collected,  finely  divided,  and 
saponified  with  an  alcoholic  solution  of  sodium  ethylate  in  the  cold  accord- 
ing to  Baisch's  method ,2  and  the  various  carbohydrates  separated  according 
to  his  suggestion. 

To  the  physician,  who  naturally  wants  simple  and  quick  methods,  the 
bismuth  test  is  especially  to  be  recommended.  If  this  test  gi\-es  negative 
results,  the  urine  is  to  be  considered  as  free  from  sugar  in  a  clinical  sense. 
If  it  gives  positive  results,  the  presence  of  sugar  must  be  controlled  by 
other  tests,  especially  by  the  fermentation  test. 

Other  tests  for  sugar,  as,  for  example,  the  reaction  with  orthonitrophenyl- 
propiolic  acid,  picric  acid,  diazobenzeiie-sulphonic  acid,  are  superfluous.  The 
reaction  with  n-naphthol,  which  is  a  reaction  for  carbohydrates  in  general,  for 
glucuronic  acid  and  mucin,  may,  because  of  its  extreme  delicac}',  give  rise  to 
mistakes,  and  is  therefore  not  to  be  recommended  to  physicians.  Normal  urines 
give  this  test,  and  if  the  strongly  diluted  urine  gives  the  reaction  the  presence 
may  be  suspected  of  great  quantities  of  carbohydrates.  In  these  cases  more 
positive  results  are  obtained  by  using  other  tests.  This  test  requires  great  clean- 
liness, and  it  has  the  inconvenience  that  sufficiently  pure  sulphuric  acid  is  not 
always  readily  procurable.  Several  investigators,  such  as  v.  Udraxsky,  Luther, 
Roos  and  Treupel,^  have  investigated  this  test  in  regard  to  its  applicabilit}^ 
as  an  approximate  test  for  carbohydi-ates  in  the  luine. 

Quantitative  Determination  of  Sugar  in  the  Urine.  The  urine  for  such 
an  estimation  must  fisrt  be  tested  for  proteid,  and  if  any  be  present  it  must 
l^e  removed  by  coagulation  and  the  addition  of  acetic  acid,  care  l^eina:  taken 

'  Pfliiger's  Arch  .  91. 

^  Zeitschr.  f.  physiol.  Chem.,  19. 

^  See  Roos  and  Treupel,  Zeitschr.  f.  phj-siol.  Chem.,  15  and  16. 


660  URINE. 

not  to  increase  or  diminish  the  original  volume  of  urine.  The  quantity  of 
sugar  may  be  determined  by  titration'  with  Fehlixg's  or  Knapp's  solu- 
toin.  by  fermextatiox,  by  polarizatiox,  and  in  other  ways. 

The  titration  liquids  not  only  react  with  sugar,  but  also  with  certain 
other  reducing  substances,  and  on  this  account  the  titration  methods  give 
rather  high  results.  When  large  quantities  of  sugar  are  present,  as  in  typi- 
cal diabetic  urine,  which  generally  contains  a  lower  percentage  of  normal 
reducing  constituents,  this  is  indeed  of  little  account;  but  when  small  quan- 
tities of  sugar  are  present  in  an  othenvise  normal  urine,  the  mistake  may, 
on  the  contrar\%  be  important,  as  the  reducing  power  of  normal  urine  may 
correspond  to  5  p.  m.  dextrose  (see  page  609).  In  such  cases  the  titration 
procedure  must  be  employed  in  connection  with  the  fermentation  method, 
which  will  be  described  later.  It  is  to  be  remarked  that  in  typical  diabetic 
urines  with  considerable  quantities  of  sugar  the  titration  with  Fehlixg's 
solution  is  just  as  reliable  as  with  Knapp's  solution.  When  the  urine  on 
the  other  hand,  contains  only  little  sugar  with  normal  amounts  of  physiolog- 
ical constituents,  then  the  titration  with  Feeling's  solution  is  more  difficult, 
in  certain  cases  indeed  almost  impossible,  and  the  results  become  ver\^ 
uncertain.  In  such  cases  Knapp's  method  gives  good  results,  according  to 
Worm  Muller  and  his  pupils.^ 

The  titration  with  Fehling's  solution  depends  on  the  power  of  sugar 
to  reduce  copper  oxide  in  alkaline  solutions.  For  this  there  was  formerly 
employed  a  solution  which  contained  a  mixture  of  copper  sulphate,  Rochelle 
salt,  and  sodium  or  potassium  hydrate  (Fehling's  solution) ;  but  as  such 
a  solution  readily  changes,  use  is  made  of  a  copper-sulphate  solution  and 
an  alkaline  Rochelle-salt  solution  prepared  separateh^  and  the  two  solutions 
mixed  in  equal  volumes  before  using. 

The  concentration  of  the  copper-sulphate  solution  is  such  that  10  c.c.  of 
this  solution  is  reduced  by  0.0.5  gram  of  dextrose.  The  copper-sulphate 
solution  contains  34.6.5  grams  of  pure,  cr}\stanized,  non-efflorescent  copper 
sulphate  in  1  liter.  The  sulphate  is  crj^stalUzed  from  a  hot  saturated  solu- 
tion by  cooling  and  stirring,  and  the  crj^stals  are  separated  from  the  mother- 
liquor  and  pressed  between  blotting-paper  until  dr\^  The  Rochelle-salt 
solution  is  prepared  by  dissolving  173  grams  of  the  salt  in  350  c.c.  of  water, 
adding  600  c.c.  of  a  caustic-soda  sohition  of  a  specific  gravity  of  1.12,  and 
diluting  with  water  to  1  liter.  According  to  Worm  AIullfr,  these  three 
liquids — Rochelle-salt  solution,  caustic  soda,  and  water — should  be  sepa- 
rately boiled  before  mixing  together.  For  each  titration  mix  in  a  small  flask 
or  porcelain  dish  exactly  10  c.c.  of  the  copper-sulphate  solution  and  10 
c.c.  of  the  alkaline  Rochelle-salt  solution  and  add  30  c.c.  of  water. 

The  urine,  freed  from  proteid,  is  diluted  with  water  before  the  titration, 
so  that  10  c.c.  of  the  copper  solution  requires  between  5  and  10  c.c.  of 
the  diluted  urine,  which  corresponds  to  between  1  per  cent  and  J  per  cent 
of  sugar.  A  urine  of  a  specific  gravity  of  1.030  may  be  diluted  five  times; 
on3  more  concentrated,  ten  times.  The  urine  so  diluted  is  poured  into  a 
burette  and  allowed  to  flow  into  the  boiling  copper-sulphate  and  Rochelle- 
salt  solution  until  the  copper  oxide  Is  completely  reduced.  This  has  taken 
place  when,  immediately  after  boiling,  the  blue  color  of  the  solution  disap- 
pears.    It  is  very  difficult  and  requires  some  practice  to  exactly  determine 

>  Pfliiger's  Arch.,  16  and  23;   Otto,  Journal  f.  prakt.  Chem.  (N.  F.),  26. 


SUGAR  IX  UPJXE.  6G1 

this  point,  especially  when  the  copper  suboxide  settles  with  difficulty.  To 
determine  whether  the  color  has  disappeared,  allow  the  copper  suboxide 
to  settle  a  little  below  the  meniscus  formed  l^y  the  surface  of  the  liquid. 
If  this  layer  is  not  h\\ie,  the  operation  is  repeated,  adding  0.1  c.c.  less  of 
urine;  and  if,  after  the  copper  suboxide  has  settled,  the  liquid  has  a  blue 
color,  the  titration  may  be  considered  as  completed.  Because  of  the  diffi- 
culty in  oljtaining  tliis  point  exactly  another  end-reaction  has  been  sug- 
gested. Tliis  consists  in  filtering  immediately  after  boiling  a  small  portion 
of  the  titrated  mixture  through  a  small  filter  into  a  test-tube  wMch  con- 
tains a  little  acetic  acid  and  a  few  drops  of  potassium-ferrocyanide  solution 
and  water.  The  smallest  quantity  of  copper  is  shown  by  a  red  coloration. 
If  the  operation  is  quickly  conducted  so  that  no  oxidation  of  the  suboxide 
into  oxide  takes  place,  this  end-reaction  is  of  value  for  urines  which  are  rich 
in  sugar  and  poor  in  urea  and  which  have  been  strongly  diluted  with  water. 
In  urines  poor  in  sugar  which  contain  the  normal  amount  of  urea  and  which 
have  not  been  considerably  diluted,  a  considerable  quantity  of  ammonia 
may  be  formed  from  the  urea  on  boiling  the  alkaUne  liquid.  This  ammonia 
dissolves  the  suboxide  in  part,  which  then  easily  passes  into  oxide;  besides 
the  dissolved  suboxide  gives  a  red  color  with  potassium  ferrocyanide.  In 
just  those  cases  in  which  the  titration  is  most  difficult  this  end-reaction 
is  the  least  reUable.  Practice  also  renders  it  unnecessary,  and  it  is 
therefore  best  to  depend  simply  upon  the  appearance  of  the  liquid. 

To  facilitate  the  settUng  of  the  copper  suboxide  and  therebv  clearing 
the  liquid,  Munk  ^  has  suggested  the  addition  of  a  little  calcium-chloride 
solution  and  boiUng  again.  A  precipitate  of  calcium  tartrate  is  produced 
which  carries  down  the  suspended  copper  suboxide  with  it.  and  the  color  of 
the  liquid  can  then  be  seen  more  readily.  This  artifice  succeeds  in  many 
cases,  but  unfortunately  there  are  urines  in  which  the  titration  wtih  Fehling's 
solution  in  no  way  gives  exact  results.  In  those  cases  in  which  onlv  small 
quantities  of  sugar  exist  in  a  urine  rich  in  physiological  constituents  it  is 
best  to  dissolve  a  ver}-  exactly  weighed  quantity  of  pure  dextrose  or  dex- 
trose-sodium chloride  in  the  urine.  The  urine  can  now  be  stronglv  diluted 
with  water"  and  the  titration  l^ecomes  successful.  The  difference'  between 
the  sugar  added  and  that  found  by  titration  gives  the  reducing  power  of  the 
original  urine  calculated  as  dextrose. 

The  necessarj^  conditions  for  the  success  of  the  titration  under  all  cir- 
cumstances are,  according  to  Soxhlet,^  the  following:  The  copper-sul- 
phate and  Rochelle-salt  soluti^^n  must,  as  above,  be  diluted  to  50  c.e.  with, 
water;  the  urine  should  contain  only  between  0.5  and  1  per  cent  of  sugar, 
and  the  total  quantity  of  urine  required  for  the  reduction  must  be  added 
to  the  titration  liquid  at  once  and  boiled  with  it.  From  this  last  con- 
dition it  follows  that  the  titration  is  dependent  upon  minute  details,  and 
several  titrations  are  required  for  each  determination. 

It  is  best  to  give  here  an  example  of  the  titration.  The  proper  amount 
of  copper-sulphate  and  Rochelle-salt  solution  and  water  (total  volume  =  50 
c.c.)  is  heatecl  to  boiling  in  a  flask;  the  color  must  remain  blue.  The  urine 
diluted  five  times  is  now  added  to  the  boiling-hot  liquid.  1  c.c.  at  a  time; 
after  each  addition  of  urine  boil  for  a  few  seconds  and  look  for  the  appear- 
ance of  the  end-reaction.     If  one  finds,  for  example,  that  3  c.c.  is  too  little, 


*  Virchow's  Arch.,  105.  ^  Journ.  f.  prakt.  Chem.  (X.  F.),  21. 


662  URINE. 

but  that  4  c.c.  is  too  much  (the  Uquid  becoming  yellowish),  then  the  urine 
has  not  been  sufficiently  diluted,  for  it  should  require  between  5  and  10  cc. 
of  the  urine  to  produce  the  complete  reduction.  The  urine  is  now  diluted 
ten  times,  and  it  should  require  between  6  and  8  c.c.  for  a  total  reduction. 
Now  prepare  four  new  tests,  which  are  boiled  simultaneously  to  save  time, 
and  add  at  one  time  respectively  6,  6^,  7,  and  7^  c.c.  of  urine.  If  it  is 
found  that  between  6^  and  7  c.c.  are  necessary  to  produce  the  end-reac- 
tion, then  make  four  other  tests,  to  which  add  respectively  6.6,  6.7,  6.8, 
and  6.9  c.c.  of  urine.  If  in  this  case  the  liquid  is  still  somewhat  bluish 
with  6.7  c.c.  and  completely  decolorized  with  6.8  c.c,  the  average  figure 
6.75  c.c.  is  considered  as  correct. 

The  calculation  is  simple.  The  6.75  c.c.  used  contains  0.05  gram  of 
sugar,  and  the  percentage  of  sugar  in  the  dilute  urine  is  therefore  (6.75 :0.05  = 

5 
100:a-)  =  — =  =  0.74.     But  as  the  urine  was  diluted  with  ten  times  its  vol- 
6.75 

5X  10 
ume  of  water,  the  undiluted  urine  contained     ^  „^  =7.4  per  cent.     The 
'  0.75 

general  formula  on  using  10  c.c.  of  copper-sulphate  solution  is  therefore 

5  X  w 

— ; — ,  in  which  n  represents  the  number  of  times  the  urine  has  been  diluted, 

k 
and  k  the  number  of  cubic  centimeters  of  the  diluted  urine  employed  for 
the  titration. 

The  TITRATION  ACCORDING  TO  Knapp  depends  on  the  fact  that  mercuric 
cyanide  in  alkaline  solution  is  reduced  to  metallic  mercury  by  dextrose. 
The  titration  liquid  should  contain  10  grams  of  chemically  pure  dry  mer- 
curic cyanide  and  100  c.c.  of  caustic-soda  solution  of  a  specific  gravity 
of  1.145  per  liter.  When  the  titration  is  performed  as  described  below  (ac- 
cording to  WoRM-MiJLLER  and  Otto),  20  c.c.  of  this  solution  should  cor- 
respond to  exactly  0.05  gram  of  dextrose.  If  the  process  is  carried  out  in 
other  ways,  the  value  of  the  solution  is  different. 

In  this  titration,  also,  the  quantity  of  sugar  in  the  urine  should  be 
between  ^  and  1  per  cent  and  the  extent  of  dilution  necessary^  be  de- 
termined by  a  preliminary  test.  To  determine  the  end-reaction  as  de- 
scribed below,  the  test  for  the  excess  of  mercury  is  made  with  sulphuretted 
hydrogen. 

In  performing  the  titration  allow  20  c.c.  of  Knapp's  solution  to  flow 
into  a  flask  and  dilute  vvith  80  c.c.  of  water,  or  when  the  urine  contains 
less  than  0.5  per  cent  of  sugar  use  only  40-60  c.c.  After  this  heat  to 
boiling  and  allow  the  dilute  urine  to  flow  gradually  into  the  hot  solution, 
at  first  2  c.c,  then  1  c.c,  then  0.5  c.c,  then  0.2  c.c,  and  lastly  0.1  c.c. 
After  each  addition  let  it  boil  h  minute.  When  the  end-reaction  is  approach- 
ing, the  liquid  begins  to  clarify  and  the  mercury  separates  with  the  phos- 
phates. The  end-reaction  is  determined  by  taking  a  drop  of  the  upper 
layer  of  the  liquid  into  a  capillary  tube  and  then  blowing  it  out  on  pure 
white  filter-paper.  The  moist  spot  is  first  held  over  a  bottle  containing 
fuming  hydrochloric  acid  and  then  over  strong  sulphuretted  hydrogen. 
The  presence  of  a  minimum  quantity  of  mercury  salt  in  the  liquid  is  shown 
by  the  spot  becoming  yellowish,  which  is  best  seen  when  it  is  compared 
"with  a  second  spot  that  has  not  been  exposed  to  the  gas.  The  end-reaction 
is  still  clearer  when  a  small  part  of  the  liquid  is  filtered,  acidified  with  acetic 


SUGAR    Ix\   URINE.  663 

acid,  and  tested  with  sulphuretted  hydrogen  (OttoI).  The  calculations 
are  just  as  simple  as  for  the  previous  method. 

This  titration,  unlike  the  previous  one,  may  be  performed  equally  well 
by  daylight  and  by  artificial  light.  Knapp'.s  method  has  the  following 
advantages  over  Fehling's  method:  It  is  applicable  even  when  the  quantity 
of  sugar  in  the  urine  is  very^  small  and  that  of  the  other  urinary  constituents 
is  normal.  It  is  more  easily  performed,  and  the  titration  liquids  may  be 
kept  without  decomposing  for  a  long  time  (Worm  MiIllp^r  and  his  pupils  2). 
The  views  of  the  various  investigators  on  the  value  of  this  titration  method 
are  nevertheless  somewhat  contradictoiy. 

The  titration  according  to  Pavy  consists  in  adding  a  boiling  ammoniacal 
solution  of  copper  sulphate  to  the  urine  until  it  is  decolorized,  when  the 
suboxide  formed  is  dissolved  by  the  ammonia  into  a  colorless  solution. 
The  admission  of  air  must  be  completely  excluded.  In  regard  to  the  per- 
formance of  this  highly  recommended  method  we  must  refer  to  the  works 
of  Pavy,  Kumagawa  and  Suto,  and  Sahli.^ 

Besides  the  above-described  methods  there  are  various  others.  K.  B. 
Lehmann  uses  an  excess  of  copper  salt  and  retitrates  with  potassium  iodide 
and  hyposulphite.  The  sugar  can  also  be  determined  according  to  Allihn, 
and  especially  according  to  Pfluger's  modification  of  this  method."* 

Estimation  of  the  Quantity  of  Sugar  by  Fermentation.  This 
may  be  done  in  various  ways;  the  simplest  method,  and  one  at  the  same 
time  sufficiently  exact  for  ordinaiy  cases,  is  that  of  Roberts.  This  con- 
sists in  determining  the  specific  gravity  of  the  urine  before  and  after  fer- 
mentation. In  the  fermentation  of  sugar,  carbon  dioxide  and  alcohol  are 
formed  as  chief  products  and  the  specific  gravity  is  lowered,  partly  on 
account  of  the  disappearance  of  the  sugar  and  partly  on  account  of  the 
production  of  alcohol.  Roberts  found  that  a  decrease  of  0.001  in  the 
specific  gravity  corresponded  to  0.23  per  cent  sugar,  and  this  has  been  sub- 
stantiated since  by  several  other  investigators  (Worm  ^Iuller  and  others). 
If  the  urine,  for  example,  has  a  specific  gravity  of  1.030  before  fermentation 
and  l.OOS  after,  then  tlie  quantity  of  sugar  contained  therein  was  22X0.23 
=  .5.06  per  cent. 

In  performing  this  test  the  specific  gra\dty  must  be  taken  at  the  same 
temjjerature  before  and  after  the  fermentation.  The  urine  must  be  faintly 
acid,  and  when  necessarv^  it  should  be  acidified  with  a  Httle  hydrochloric 
acid  or  sulphuric  acid.  The  activity  of  the  yeast  must,  when  necessar\',  be 
controlled  by  a  special  test.  Place  200  c.c.  of  the  urine  in  a  400  c.c.  flask, 
add  a  piece  of  compressed  yeast  the  size  of  a  pea.  and  subdi^^de  the  veast 
through  the  liquid  by  shaking;  close  the  flask  with  a  stopper  provided  with 
a  finely -drawn-out  glass  tube,  and  allow  the  test  to  stand  at  the  temperature 
of  the  room  or.  still  better,  at  30-35'""  C.  After  24  hours  the  fermenta- 
tion is  ordinarily  ended,  but  this  must  be  verified  by  the  bismuth  test. 
After  complete  fermentation  filter  through  a  drv^  filter,  bring  the  filtrate  to 
Ihe  proper  temperature,  and  determine  the  specific  gravitv. 

'  Journal  f.  prakt.  Chem.,  26. 

2  Pfluger's  Arch.,  16  and  23. 

'  Favy,  The  Physiology  of  the  Carbohydrates,  London,  1894;  Kumagawa  and 
Suto,  Salkowski's  Festschrift,  1904;  SahH,  Deutsch.  med.  Wochenschr.,  1905.  In 
regard  to  other  methods,  see  Huppert-Ne'ubauer.  Analyse  des  Harnes. 

*  Lehmann,  Arch.  f.  Hygiene,  30;  Pfliiger,  Pfluger's  Arch.,  66. 


664  URINE. 

If  the  specific  gravity  l^e  determined  with  a  good  pyknometer  supplied 
with  a  thermometer  and  an  expansion-tube,  this  method,  when  the  quan- 
tity of  sugar  is  not  less  than  4-5  p.  m.,  gives,  according  to  ^\  orm  Duller. 
very  exact  results,  but  this  has  been  disputed  by  Budde.^  For  the  physi- 
cian the  method  in  this  form  is  not  quite  serviceable.  Even  when  the 
specific  gravity  is  determined  by  a  delicate  urinometer  which  can  give  the 
density  to  the  fourth  decimal,  quite  exact  results  are  not  obtained,  because 
of  the  ordinary  errors  of  the  method  (Buddk);  but  the  errors  are  usually 
smaller  than  those  which  occur  in  titrations  made  by  unskilled  hands. 

When  the  c^uantity  of  sugar  is  less  than  .5  p.  m.  these  methods  cannot 
be  used.  Such  small  amounts  cannot,  as  already  mentioned,  be  determined 
by  titration  directly,  because  the  reducing  power  of  normal  urine  corre- 
sponds to  4-5  p.  m.  of  sugar.  In  such  cases,  according  to  Worm  ^Julij:r, 
it  is  better  first  to  determine  the  reduction  power  of  the  urine  by  titration 
with  Knapp's  solution,  then  ferment  the  urine  with  the  addition  of  yeast 
and  titrate  again  with  Knapp's  solution.  The  difference  found  between 
the  two  titrations  calculated  as  sugar  gives  the  true  quantity  of  the  latter. 

The  determination  of  the  sugar  by  fermentation  can  be  so  performed 
that  the  loss  in  weight  due  to  the  CO2  can  be  estimated  or  the  volume  of 
the  gas  measured.  For  this  last  purjDOse  Lohxsteix^  has  constructed  a 
special  fermentation  saccharometer,  of  which  his  "precision  saccharometer'' 
is  to  be  recommended.  Based  upon  Lohxstein's  instrument,  Wagner  ^^ 
has  constructed  a  '  fermentation  saccharo-mano meter,"  which  has  certain 
advantages  over  Lohnstein's  apparatus. 

Estimation  of  Sugar  by  Polarization.  In  this  method  the  urine 
must  be  clear,  not  too  deeply  colored,  and,  above  all,  must  not  contain 
any  other  optically  active  substances  besides  dextrose.  The  urine  may 
contain  several  levorotatory^  substances  such  as  proteids,  5-oxybutyric 
acid,  conjugated  glucuronic  acids,  the  so-called  Leo's  sugar,  and  less  often 
cystine,  all  of  which  areunfermentable.  The  proteid  is  removed  by  coagu- 
lation, and  the  others  are  detected  by  the  polariscope  after  complete  fer- 
mentation. The  fermentable  levulose  is  detected  in  a  special  manner 
(see  below),  and  the  dextrorotatory  milk-sugar  differs  from  dextrose  in  its 
not  fermenting  readily.  By  using  a  delicate  instrument  and  with  suffi- 
cient practice  very  exact  results  can  be  ol:)tained  by  this  method.  The 
value  of  this  procedure  consists  in  the  rapidity  with  which  the  determina- 
tion can  l)e  made.  In  using  instruments  specially  constiTicted  for  clinical 
purposes  the  accuracy  is  less  than  with  the  less  expensive  fermentation 
test.  Un^er  such  circumstances,  and  as  the  estimation  by  means  of  polari- 
zation can  he  performed  with  exactitude  only  by  specially  trained  chemists, 
it  is  hardly  worth  while  to  give  this  method  in  detail,  and  the  reader  is 
referred  to  handbooks  for  hints  in  the  use  of  the  apparatus. 

Levulose.  Levog^^rate  urines  containing  sugar  have  been  observed  bv 
several  ol)servers.  although  the  nature  of  the  sugar  was  not  well  known  to 
the  earlier  oljservers.      In   recent  years  several  positively  authentic  cases 

>  Roberts,  Edinburgli  Med.  Joum.,  18G1,  and  The  Lancet,  1,  1862;  Worm-Miiller, 
Pfliiger's  Arch.,  3S  and  37;  Budde,  ibid.,  40,  and  Zeitschr.  f.  physiol.  Chem  ,  13  See 
also  Huppert-Neubauer,  10.  Aufl.,  and  Lohnstein,  Pfliiger's  Arch.,  62. 

^Berlin,  klin.  Wochenschr.,  3o,  and  Allg.  med    Central-Ztg ,  1899. 

3  Miinch.  med.  Wochen.schr. ,  190.5. 


LACTOSE.  665 

of  le"VTilosiiria  have  been  described,  and  also  cases  of  diabetes  have  been 
found  where  levulose  exists  in  the  urine  besides  dextrose. 

Le\'ulose  may  be  detected  as  follows:  The  urine  is  levorotatorj-  and 
the  levorotatory  substance  ferments  with  yeast.  The  urine  gives  the  ordi- 
nary reduction  tests  and  the  ordinar}-  phenylglucosazone.  With  methyl- 
phem-lhydrazine  it  gives  the  characteristic  le\-ulose  methylphenvlosazone, 
and  it  also  gives  Seliwaxoff's  reaction  on  heating  after  the  addition  of 
an  equal  volume  of  hydrochloric  acid  and  a  Httle  resorcin.  With  this  test 
it  must  be  remarked  that  too  lengthy  or  too  strong  heating  must  not  be 
applied,  since  other  carbohydrates  may  also  give  the  reaction  (see  page 
119  and  the  works  of  Rosix  and  Umber).  After  heating  and  cooling  it 
can  be  neutralized  with  soda  and  shaken  out  with  amyl  alcohol.  This 
removes  a  red  pigment  which  gives  a  band  in  the  spectrum  tetween  E  and  h 
on  stronger  concentration;  also  a  band  in  the  blue  at  F  (Rosix).i 

Laiose  is  a  substance  named  by  Huppert  and  found  by  Leo  •  in  diabetic  urines 
in  certain  cases,  and  which  he  considers  as  a  sugar.  It  is  levogjTate,  amorphous, 
and  does  not  taste  sweet,  but  rather  sharp  and  salty.  Laiose  has  a  reducing 
action  on  metallic  oxides,  does  not  ferment,  and  gives  a  non-crystalline,  yellowish- 
brown  oil  with  phenylhydrazine.  There  is  no  positive  proof  as  yet  that  this 
substance  is  a  sugar. 

Lactose.  The  appearance  of  lactose  in  the  urine  of  pregnant  women 
was  first  shown  by  the  observations  of  De  Sixety  and  F.  Hofmeistek. 
and  this  has  been  substantiated  by  other  investigators.  After  the  ingestion 
of  large  quantities  of  milk-sugar  some  lactose  may  be  found  in  the  urine 
(see  Chapter  IX  on  absorption).  Laxgsteix  and  Steixitz  have  observed 
the  passage  of  lactose  and  also  of  galactose  ^  into  the  urine  of  nurslinos 
with  diseases  of  the  stomach.  The  passage  of  lactose  into  the  urine  is 
called  lactosuria. 

The  positive  detection  of  this  sugar  in  the  urine  is  difficult,  because 
it  is,  like  dextrose,  dextrogA-rate  and  also  gives  the  usual  reduction  tests. 
If  urine  contains  a  dextrog}'rate,  non-fermentable  sugar  which  reduces 
bismuth  solutions,  then  it  Is  ver\'  probable  that  it  contains  lactose.  It 
must  be  remarked  that  the  fermentation  test  for  lactose  is.  according  to 
the  experience  of  Lu.sk  and  ^'oit,-^  best  performed  by  using  pure  cultivated 
yeast  (saccharomyces  apiculatus).  This  yeast  only  ferments  tl^e  dextrose, 
while  it  does  not  decompose  the  milk-susar.  If,  according  to  Voir.  Rub- 
ner's  test  is  performed  without  heating  to  boiling,  but  only  to  80°  C,  the 
color  becomes  yellow  or  brown  in  the  presence  of  lactose,  instead  of  red. 
The  most  positive  means  for  the  detection  of  this  sugar  is  to  isolate  the 

•  Umber,  Salkowski's  Festschrift,  Berlin,  1904;  Rosin,  ibid.,  and  Zeitsclir.  f.  physiol. 
Chem.,  38. 

'  Vircliow's  Arch.,  107. 

3  Hofmeister,  Zeitschr.  f.  physiol.  Chem.,  1,  which  also  contains  the  pertinent 
literature.     See  also  Lemaire,  ibid.,  21 :  Langstein  and  Steinitz,  Hofniei.ster's  Beitrage,  7. 

*  Carl  Voit,  Ueber  Die  Glycogenbildung  nach  Aufnahme  verschiedener  Zuckeraten, 
Zeitsclir.  f.  Biologie,  28. 


666  URINE. 

sugar  from  the  urine.     This  may  be  done  by  the  following  method,  suggested 
by  F.  Hofmeister: 

Precipitate  the  urine  with  sugar  of  lead,  filter,  wash  with  water,  unite  the 
filtrate  and  wash-water,  and  precipitate  with  ammonia.  The  liquid  filtered 
from  the  precipitate  is  again  precipitated  by  sugar  of  lead  and  ammonia  until  the 
last  filtrate  is  optically  inactive.  The  several  precipitates  with  the  exception 
of  the  first,  which  contains  no  sugar,  are  united  and  washed  with  water.  This 
precipitate  is  decomposed  in  the  cold  with  sulphuretted  hydrogen  and  filtered. 
The  excess  of  sulphuretted  hydrogen  is  driven  off  by  a  current  of  air;  the  acids 
set  free  are  removed  by  shaking  with  silver  oxide.  Now  filter,  remove  the 
soluble  silver  by  sulphuretted  hydrogen,  treat  with  barium  carbonate  to  unite 
with  any  free  acetic  acid  present,  and  concentrate.  Before  the  evaporated  residue 
becomes  syrupy  it  is  treated  with  90  per  cent  alcohol  until  a  flocculent  precipi- 
tate is  formed  which  settles  quickly.  The  filtrate  from  this  when  placed  in  a 
desiccator  deposits  crystals  of  lactose,  which  are  purified  by  recrystallization, 
■decolorizing  with  animal  charcoal  and  boiling  with  60-70  per  cent  alcohol. 

Pentoses.  Salkowski  and  Jastrowitz  first  found  in  the  urine  of  per- 
sons addicted  to  the  morphine  habit  a  variety  of  sugar  which  was  a  pen- 
tose and  yielded  an  osazone  which  melted  at  159°  C.  Since  this  several 
other  cases  of  pentosuria  have  been  observed,  and  according  to  Kulz  and 
VoGEL  small  amounts  of  pentose  also  occur  in  the  urine  of  diabetics,  as 
also  in  the  urine  of  dogs  with  pancreatic  or  phlorhizin  diabetes.^ 

The  pentose  isolated  by  Neuberg  from  the  urine  in  chronic  pentosuria 
was  i-arabinose.  In  alimentar}^  pentosuria  the  /-arabinose  of  the  plant 
food  may  be  found  in  the  urine.  The  appearance  of  pentoses  in  the  urine 
after  eating  fruits  and  fruit-juices  has  been  repeatedly  observed  by  Blumen- 
THAL  and  also  by  v.  Jaksch.^ 

A  urine  containing  pentose  reduces  bismuth  as  well  as  copper  solutions^ 
although  the  reduction  is  not  so  rapid,  but  appears  gradually.  If  only 
pentose  is  present,  the  urine  does  not  ferment,  but  in  the  presence  of  dex- 
trose small  amounts  of  pentose  may  also  undergo  fermentation.  The 
preparation  of  the  osazone  serves  in  the  detection  of  pentoses;  this  com- 
pound when  pure  melts  at  166-168°  C,  but  when  obtained  from  the  urine 
has  a  melting-point  of  156-160°  C.  The  phloroglucin  or  orcin  tests  can  also 
l3e  employed  (see  page  111).  Of  these  the  last  is  most  preferable,  especially 
as  it  excludes  a  confusion  with  the  conjugated  glucuronic  acids. 

The  orcin  test  can  be  performed  as  follows:  5  c.c.  of  the  urine  is  mixed 
with  an  equal  volume  of  HCl  sp.  gr.  1.19,  a  small  amount  of  orcin  added  and 
the  whole  heated  to  boiling.  As  soon  as  a  greenish  cloudiness  appears, 
cool  the  mixture  off  and  shake  carefully  with  amyl  alcohol  The  amyl- 
alcohol  solution  is  used  in  the  spectroscopic  examination.  The  precipita- 
tion of  a  bluish-green  pigment  is  in  itself  significant. 


'In  regard    to  the  literature,  see  foot-note   1,  page   110.      See  also  Bkmienthal, 
"Die  Pentosurie,"  Deutsche  IClinik,  1902. 

»  Blumenthal,  Deutsche  KUnik,  1902;  v.  Jaksch,  Centralbl.  f  innere  Medizin,  1906. 


CONJUGATED   GLUCURONIC   ACIDS.  667 

BiAL  1  uses  as  reagent  30  }3er  cent  hydrochloric  acid,  which  contains 
1  gram  of  orcin  and  25  drops  of  a  ferric-chloride  solution  (62.9  per  cent  of 
the  crj-stalline  salt)  in  500  c.c.  of  the  acid.  4-5  c.c.  of  the  reagent  is 
heated  to  boiling  and  then  a  few  drops  (not  more  than  1  c.c.)  of  the  urine 
is  added  to  the  hot  but  not  boiling  liquid.  In  the  presence  of  pentose  the 
Hquid  turns  a  beautiful  green.  The  usefulness  of  Bial's  reagent  is  ques- 
tioned by  several  experimenters.  The  delicacy  is  not  ver^-  great  and  the 
possibility  of  confounding  with  other  carbohydrates  is  not  excluded. 

Lepixe  and  Boulud^  have  shown  the  presence  of  maltose  in  cases  of 
diabetes.  After  boiling  with  hydrochloric  acid  the  specific  rotation  dimin- 
ishes, while  the  reducing  power  increases  in  such  urines. 

Conjugated  Glucuronic  Acids.  Certain  conjugated  glucuronic  acids 
such  as  menthol-  and  turpentine-glucuronic  acid  may  spontaneously  decom- 
pose in  the  urine,  and  in  this  case  they  may  readik  lead  to  a  confusion  vrith 
pentoses.  The  urine  should  l^e  always  as  fresh  as  possible  for  these  exami- 
nations. 

A  confusion  of  the  glucuronic  acids  which  have  a  reducing  power  on 
copper  or  bismuth  solutions  with  dextrose  and  le\'ulose  can  be  prevented 
by  the  fermentation  test.  They  may  also  be  distinguished  from  dextrose 
by  their  optical  behavior,  as  the  conjugated  glucuronic  acids  are  levo- 
g}-rate.  On  boiling  with  an  acid  dextrorotatory  glucuronic  acid  is  pro- 
duced and  the  levorotation  is  changed  to  dextrorotation. 

The  conjugated  glucuronic  acids,  hke  the  pentoses,  give  the  phloro- 
glucin-hydrochloric-acid  test.  On  the  contrar\-  they  do  not  give  the  orcin 
test  directly,  but  only  after  cleavage  with  the  setting  free  of  glucuronic 
acid.  On  using  Bial's  reagent  no  mistaking  for  pentoses  occurs,  although 
this  statement  requires  further  substantiation.  The  pentoses  may  also 
be  isolated  and  identified  by  their  osazones.  The  occurrence  of  conjugated 
glucuronic  acid  in  the  urine  is  shown  when  the  urine  does  not  give  the 
orcin-hydrochloric-acid  reaction  directly,  but  only  after  boihng  with  the 
acid.  A  further  proof  is  that  suggested  by  v.  Alfthax.^  500  c.c.  of  the 
urine  is  benzoylated  and  the  ester  obtained  saponified  with  sodium  ethylate. 
The  free  and  conjugated  glucuronic  acid  is  thus  obtained  as  sodium  com- 
pounds, insoluble  m  alcohol,  while  the  pentoses,  if  present,  remain  in  the 
alcoholic  filtrate.  We  have  no  suflBcient  experience  as  to  the  value  of  this 
method. 

The  surest  method  is  that  suggested  by  ^Mayer  and  Neuberg,^  which 
consists  in  precipitating  the  urine  with  basic  lead  acetate,  decomposing  the 
precipitate  with  H^S,  boiUng  with  dilute  sulphuric  acid  in  order  to  spUt  the 
conjugated  acid,  and  then  after  neutrahzing  A\-ith  soda  prepare  the  charac- 


*  Deutsch.  med.  Wochenschr.,  1903;  '  Compt.  rend.,  132. 

5  Arch.  f.  exp.  Path.  u.  Pharm.,  4",  *  Zeitschr.  f.  physiol.  Chem.,  29. 


6G8  URINE. 

teristic  bromphenylhydrazine  compound  of  glucuronic  acid  (see  page  123) 
with  p-bromphenylhydrazine  hydrochloride  and  sodium  acetate. 

Inosite  occurs  in  the  urine  in  albuminuria  and  in  diabetes  mellitus,  but 
only  rarely  and  in  small  quantities.  Inosite  is  also  found  in  the  urine  after 
the  excessive  drinking  of  water.  According  to  Hoppe-Seyler  ^  traces  of 
inosite  occur  in  all  normal  urines. 

In  detecting  inosite  the  proteid  is  first  removed  from  the  urme.  Then  con- 
centrate the  urine  on  the  water-bath  to  j  of  its  original  volume  and  precipitate 
with  sugar  of  lead.  The  filtrate  is  warmed  and  treated  with  basic  lead  acetate  as 
long  as  a  precipitate  is  formed.  The  precipitate  formed  after  twenty-four  hours 
is  washed  with  water,  suspended  in  water,  and  decomposed  with  sulphuretted 
hydrogen.  A  little  uric  acid  may  separate  from  the  filtrate  after  a  short  time. 
The  liquid  is  filtered,  concentrated  to  a  syrupy  consistency,  and  treated  while 
boiling  with  3-4  vols,  alcohol.  The  precipitate  is  quickly  separated.  After 
the  addition  of  ether  to  the  cooled  filtrate,  crystals  separate  after  a  time,  and 
these  are  purified  by  decolorization  and  recrystallization.  With  these  crystals 
perform  the  tests  mentioned  on  page  459. 

Acetone  Bodies  (acetone,  acetoacetic  acid,  /9-oxy butyric  acid).  These 
bodies,  whose  occurrence  in  the  urine  and  formation  in  the  organism  have 
been  the  subject  of  numerous  investigations,  occur  in  the  urine  especially 
in  diabetes  mellitus,  but  also  in  many  other  diseases.^  According  to  v. 
Jaksch  and  others  acetone  is  a  normal  urinary  constituent,  though  it  may 
occur  only  in  very  small  amounts  (0.01  gram  in  twenty -four  hours). 

In  regard  to  the  origin  of  these  bodies  it  was  previously  considered  that 
they  were  produced  by  an  increased  destruction  of  protein.  One  of  the 
various  reasons  for  this  was  the  increase  in  the  eUmination  of  acetone  and 
acetoacteic  acid  during  inanition  (v.  Jaksch,  Fr.  Muller^).  This  stands 
also  in  good  accord  with  the  observations  that  a  considerable  increase  in 
the  quantity  of  acetone  and  acetoacetic  acid  eliminated  is  observed  in 
such  diseases  as  fevers,  diabetes,  digestive  disturbances,  mental  diseases 
with  abstinence  and  cachexia,  where  the  body  protein  is  largely  destroyed. 
The  formation  of  acetone  bodies  from  protein  is  also  indicated  by  the  fact 
that  acetone  has  been  obtained  as  an  oxidation  product  from  gelatine  and 
protein  (Blumenthal  and  Neuberg,  Orgler*).  On  the  other  hand,  no 
parallelism  exists  between  the  acetone  bodies  and  the  nitrogen  excretion  in 

'  Handbuch  d.  physiol.  u.  pathol.  chem.  Analyse,  6.  Aufl.,  196. 

'  In  regard  to  the  extensive  literature  on  acetone  bodies  the  reader  is  referred  to 
Huppert-Neubauer,  Ham-Analyse,  10.  Aufl.,  and  v.  Noorden's  Lehrb.  d.  Pathol,  des 
Stoffwechsels.     Berlin,  1906. 

^  V.  Jaksch,  Ueber  Acetonurie  und  Diaceturie.  Berlin,  1885;  Fr.  Mi'iller,  Berieht 
iiber  die  Ergebnisse  des  an  Cetti  ausgefiihrten  Hungerversuches.  Berlin,  klin.  Wochen- 
schr.,  1887. 

^  Blumenthal  and  Xeuberg,  Deutsch.  med.  Wochenschr.,  1901;  Orgler,  Hofmeis- 
ter's  Beitrage,  1. 


ACETONE   BODIES.  669 

diabetics,  and  the  fact  that  in  man  no  certain  relationship  exists  between  the 
acetone  elimination  and  the  nitrogen  and  sulpliur  excretion  seem  to  show 
that  the  acetone  bodies  are  not  entirely  derived  from  the  proteins.  In 
man  the  excretion  of  acetone  does  not  increase  with  the  rise  in  the  quan- 
tity of  protein,  and  an  increase  in  the  latter  above  the  average  causes  a 
diminution  in  the  elimination  of  acetone  (Rosexfeld,  Hirschfeld,  Fr. 
YoiT^).  At  the  present  time  the  tendency  is  more  and  more  to  the  view 
that  the  acetone  bodies  do  not  originate  from  the  proteins  but  from  the 
fats;  if  they  are  not  the  only  source,  they  are  at  least  the  most  important. 

It  is  generally  accepted  that  in  man  the  carbohydrates  have  a  strong 
influence  on  the  elimination  of  acetone  bodies,  namely,  the  exclusion  of 
carbohydrates  from  the  food  or  the  diminution  in  their  amount  or  their 
assimilation  may  lead  to  more  or  less  increased  elimination  of  acetone 
bodies.  This  behavior  may  occur  in  diabetes  as  well  as  in  starvation  and 
in  the  above-mentioned  diseased  conditions.  With  abundant  supply  of 
carbohydrates  the  acetone  bodies  are  markedly  diminished  or  may  dis- 
appear entirely,  and  a  similar  retarding  action  has  been  found  by  Satta^ 
to  be  brought  about  by  other  bodies,  such  as  glycerine,  tartaric  acid,  lactic 
acid,  and  citric  acid.  The  increased  excretion  of  acetone  with  carbohydrate 
starvation  occurs  also  in  healthy  individuals  with  a  fatty  diet,  or  on  the 
supply  of  sufficient  calories  in  other  ways  (alimentar\'  acetonuria). 

If  we  do  not  accept  the  formation  of  acetone  bodies  from  proteins, 
then  we  must  admit  such  a  formation  from  the  fats.  As  proof  of  this 
there  are  certain  cases  of  diabetes  with  strong  eUmination  of  acetone  bodies 
(^-oxybutyric  acid)  where  the  quantity  of  protein  transformed  was  too 
small  to  account  for  the  acetone  bodies  OIAGxus-LE^•Y) .  The  free  elimi- 
nation of  acetone  bodies  in  starvation  may  also  depend  upon  the  fact  that 
a  great  part  of  the  body  fat  is  consumed,  and  in  several  cases  a  certain 
relationship  has  been  found  between  the  fat  consumed  and  the  acetone 
bodies  eliminated.  Certain  investigators  (Geelmuydex,  Schwarz.  Wald- 
VOGEL^)  have  also  observed  an  increase  in  the  acetonuria  on  partaking 
of  fatty  food. 

There  is  no  doubt  that  the  fats  bear  a  certain  relationship  to  the  ace- 
tone bodies,  and  that  they  are  probably  in  part  the  source  of  the  same. 
It  has  not  been  proved,  on  the  contrary,  that  the  fats  are  the  only  or  the 
most  important  source  of  the  acetone  bodies,  and  to  all  appearances  we  must 

'  Hirschfeld,  Zeitschr.  f.  klin.  Med.,  28;  Geelmuyden,  see  Maly's  Jaliresber.,  26, 
and  Zeitschr.  f.  physiol.  Chem.,  23  and  26;  Rosenfeld,  Centralbl.  f.  innere  Med.,  16; 
Voit,  Deutsch.  Arch.  f.  khn.  Med.,  66. 

'  Hofmeister's  Beitrage,  6. 

^Magnus-Levy,  Arch.  f.  exp.  Path  lu -Pharm.,  42:  Geelmuyden,  1.  c,  and  Norsk. 
Magazin  for  Laegevidenskaben,  1900,  see  also  Zeitschr  f.  physiol.  Chem.  41,  Schwarz, 
Deutsch.  Arch,  f  klin.  Med.,  1903;  Waldvogel,  Centralbl,  f.  innere  Med.,  20. 


670  URINE. 

consider  the  proteins  equally  with  the  fats  as  the  source  of  these  bodies. 
The  researches  of  Embden  and  his  coworkers  are  of  special  interest  in  this 
connection.  After  Embden  and  Kalberlah  showed  that  the  liver  was  an 
acetone-forming  organ  Embden,  Salomon  and  Schmidt  i  showed  by  experi- 
ments with  removed  livers  that  butyric  acid,  oxybutyric  acid,  leucine, 
tyrosine,  and  in  fact  all  aromatic  bodies  (such  as  tyrosine,  phenylalanine, 
phenyl-a-lactic  acid  and  homogentisic  acid)  which  contain  a  benzene 
nucleus  which  can  be  burnt  in  the  body,  may  be  transformed  into  acetone 
in  the  liver. 

In  drawing  conclusions  as  to  the  origin  of  the  acetone  bodies  it  must 
not  be  forgotten  that  the  conditions  in  man  are  distinctly  different  from 
those  in  carnivora  (Geelmuyden,  Fr.  Voit).  In  dogs  the  elimination  of 
acetone  bodies  is  not  increased  in  starvation,  but  is  reduced;  it  is  aug- 
mented with  increased  quantities  of  meat,  runs  parallel  with  the  nitrogen 
excretion   and  is  not  diminished  by  carbohydrates  (Fr.  Voit). 

/CH3 

Acetone,  CqHbO,  dimethylketone  =  CO^         ,  occurs,  as  above  stated, 

in  ver}^  small  amounts  in  normal  urine.  In  diabetes  it  may  give  a  poma- 
ceous  or  fruit  odor  to  the  urine  as  well  as  to  the  expired  air. 

Irrespective  of  the  alimentary  acetonuria  derived  from  the  food,  there 
occurs  an  increased  elimination  of  acetone,  as  above  stated,  in  many  dis- 
eases, as  also  after  nervous  lesions,  certain  intoxications,  and  after  admin- 
istration of  phlorhizin  or  extirpation  of  the  pancreas  (v.  ^^Iering  and  Min- 
kowski, Azemar2). 

Acetone  is  a  thin,  water-clear  liquid,  boiling  at  56.3°  C.  and  possessing  a 
pleasant  odor  of  tmit.  It  is  lighter  than  water,  Avith  wliich  it  mixes  in  all 
proportions,  also  with  alcohol  and  ether.  The  most  important  reactions 
for  acetone  are  the  following. 

Lieben's  Iodoform  Test.  When  a  waterj^  solution  of  acetone  is  treated 
with  alkali  and  then  with  some  iodo-potassium-iodide  solution  and  gently 
warmed  a  yellow  precipitate  of  iodoform  is  formed,  which  is  known  by  its 
odor  and  by  the  appearance  of  the  crystals  (six-sided  plates  or  stars)  under 
the  microscope.  This  reaction  is  ver}-  delicate,  but  it  is  not  characteristic" 
oi  acetone.  Gunning's  modi-fication  of  the  iodoform  test  consists  in  using 
an  alcoholic  solution  of  iodine  and  ammonia  instead  of  the  iodine  dissolved 
in  potassium  iodide  and  alkaU  hydrate.  In  this  case,  besides  iodoform,  a 
]:>lack  precipitate  of  iodide  of  nitrogen  is  formed,  but  this  gradually  dis- 
ai")pears  on  standing,  leaving  the  iodoform  visible.     This  modification  has 


'  Hofmeister's  Beitrage,  8. 

^  Az^niar,      Ac^tonurie  experimentale."      Travaux  de   physiologic,   1898  (labora- 
toiie  de  M.  le  piofesseur  E.  Hedon,  Montpellier). 


ACETO ACETIC    ACID.  671 

the  advantage  that  it  does  not  give  any  iodoform  with,  alcohol  or  aldehyde. 
On  the  other  hand,  it  is  not  quite  so  delicate,  but  still  it  detects  0.01  milli- 
gram of  acetone  in  1  c.c. 

Reynolds's  mercuric-oxide  test  is  based  on  the  power  of  acetone  to  dis- 
solve- freshly  precipitated  HgO.  A  mercuric-chloride  solution  is  precipi- 
tated by  alcoholic  caustic  potash.  To  this  add  the  liquid  to  Ije  tested, 
shake  well,  and  filter.  In  the  presence  of  acetone  the  filtrate  contains 
mercury,  which  may  be  detected  by  ammonium  sulphide.  This  test  has 
about  the  same  delicacy  as  Guxxixo's  test.  Aldehydes  also  dissolve 
appreciable  quantities  of  mercuric  oxide. 

Legal's  Sodium  Nitroprusside  Test.  If  an  acetone  solution  is  treated 
with  a  few  drops  of  a  freshly  prepared  sodium-nitroprusside  solution  and 
then  with  caustic-potash  or  soda  solution,  the  liquid  is  colored  rubv-red. 
Creatinine  gives  the  same  color;  but  if  the  mixture  is  saturated  with  acetic 
acid,  the  color  becomes  carmine  or  purplish  red  in  the  presence  of  acetone, 
but  yellow  and  then  gradually  green  and  blue  in  the  presence  of  creatinine. 
With  this  test  paracresol  responds  with  a  reddish-yellow  color,  which 
becomes  light  pink  when  acidified  with  acetic  acid  and  cannot  be  mistaken 
for  acetone.  If  ammonia  is  employed  instead  of  the  caustic  alkah  (Le 
Nobel),  the  reaction  takes  place  with  acetone  but  not  with  aldehvde. 

Pexzoldt's  indigo  test  depends  on  the  fact  that  orthonitrobenzaldehyde 
in  alkaUne  solution  with  acetone  yields  indigo.  A  warm  saturated  and  then 
cooled  solution  of  the  aldehyde  is  treated  with  the  liquid  to  be  tested  for 
acetone  and  next  with  caustic  soda.  In  the  presence  of  acetone  the  liquid 
first  becomes  yellow,  then  green,  and  lastly  indigo  separates;  and  this  mav 
be  dissolved  with  a  blue  color  by  shaking  with  chloroform.  1.6  miUigrams 
acetone  can  be  detected  bj-  this  test. 

Bela  v.  Bitto's  *  reaction  is  based  on  the  fact  than  on  adding  a  solution  of 
metadinitrobenzene  made  alkaline  with  caustic  potash  to  acetone,  a  violet-red 
color  is  produced  wMch  becomes  cherry -red  on  acidifA'ing  \\-ith  an  organic  acid  or 
metaphosphoric  acid.  Aldehyde  gives  a  similar  \'iolet-red  color  which  becomes 
yellowish  red  on  acidification.  Creatinine  does  not  give  this  reaction.  From- 
MER  -  has  suggested  the  following  method  for  detecting  acetone:  Treat  10  c.c. 
of  the  urine  ^^^th  1  gram  potassium  hydrate  and  add  10-12  drops  of  an  alkaline 
solution  of  salicyl-aldehyde.  On  warming  a  purple-red  coloration  is  obtained  in 
the  presence  of  acetone. 

CH3 

Acetoacetic    acid,    C4Hb03,    acetylacetic    acid,   diacetic   acid=A,xT    • 

COOH 

This  acid  has  not  been  observed  as  a  physiological  constituent  of  the  urine. 
It  occurs  in  the  urine  chiefly  under  the  same  conditions  as  acetone.     Like 

'  Annal.  d.  Cheni   u.  Pharm..  2()9.  -  Berlin,  kh'n.  'Wochenschr.  1905. 


672  URINE. 

acetone  the  acetoacetic  acid  occurs  often  in  children,  especially  in  high 
fevers,  acute  exanthema,  etc.  Diacetic  acid  decomposes  readily  into 
acetone.  According  to  Araki  ^  it  is  probably  produced  as  an  intermediate 
product  in  the  oxidation  of  |9-oxybutyric  acid  in  the  organism.  The  three 
bodies  appearing  in  the  urine,  acetone,  acetoacetic  acid,  and  /3-oxybutyric 
:acid,    tand  in  close  relationship  to  each  other. 

This  acid  is  a  colorless,  strongly  acid  liquid  which  mixes  with  water,  • 
alcohol,  and  ether  in  all  proportions.  On  heating  to  boihng  with  water, 
and  especially  with  acids,  this  acid  decomposes  into  carbon  dioxide  and 
acetone,  and  therefore  gives  the  above-mentioned  reactions  for  acetone. 
It  differs  from  acetone  in  that  it  gives  a  violet-red  or  brownish-red  color 
^v'ith  a  dilute  ferric-chloride  solution.  For  the  detection  of  this  acid  we 
make  use  of  the  following  reactions  w^hich  may  be  applied  directly  to  the 
urine. 

Gerhardt's  Reaction.  Treat  10-15  c.c.  of  the  urine  -^-ith  ferric-chloride 
solution  until  it  fails  to  give  a  precipitate,  filter,  and  add  some  more  ferric 
chloride.  In  the  presence  of  acetoacetic  acid  a  wine-red  color  is  obtained. 
The  color  becomes  paler  at  the  room  temperature  within  twenty-four 
hours,  but  more  quickly  on  boiling  (differing  from  saHcylic  acid,  phenol, 
sulphocyanides).  A  portion  of  the  urine  shghtly  acidified  and  boiled  does 
not  give  this  reaction  on  account  of  the  decomposition  of  the  acetoacetic 
acid. 

Arnold  and  Lipliawsky's  Reaction.  6  c.c.  of  a  solution  containing 
1  gram  of  p-arainoacetophenone  and  2  c.c.  of  concentrated  hydrochloric  acid 
in  100  c.c.  of  water  are  mixed  with  3  c.c.  of  a  1  per  cent  potassium-nitrite 
solution  and  then  treated  with  an  equal  volume  of  urine.  A  few  drops 
of  concentrated  ammonia  are  now  added  and  violently  shaken.  A  brick- 
red  coloration  is  obtained.  Then  take  10  drops  to  2  c.c.  of  this  mixture 
(according  to  the  quantity  of  acetoacetic  acid  in  the  urine),  add  15-20  c.c. 
HCl  of  sp.  gr.  1.19,  3  c.c.  of  chloroform,  and  2-4  drops  of  ferric-chloride 
Solution  and  mix  without  shaking.  In  the  presence  of  acetoacetic  acid  the 
■chloroform  is  colored  violet  or  blue  (otherwise  only  yellowish  or  faintly 
red).  This  reaction  is  more  delicate  than  the  preceding  test  and  reacts 
with  0.04  p.  m.  acetoacetic  acid.  Large  amounts  of  acetone  (but  not  the 
quantity  occurring  in  urines)  give  this  reaction  according  to  Allard.^ 

BoNDi  and  Schwarz's^  Reaction.  5  c.c.  of  the  urine  is  titrated  drop, 
by  drop  with  iodine-potassium  iodide  solution  until  the  color  is  orange-red 
Then  w'arm  gently  and  when  the  orange-red  color  has  disappeared  add  the 

'  Zeitschr.  f.  physiol.  Chem.,  18. 

=>  Arnold,  Wien.  klin.    Wochenschr.,  1899,  and  Centralbl.  f.  innere    Med.,    1900; 
Lipliawsky,  Deutsch.  med.  Wochenschr.,  1901;  Allard,  Berl.  klin.  Wochenschr.,  1901. 
^Wien.  kiln.  Wochenschr.,  1906. 


/?-OXYBUTYRIC   ACID.  673 

iodine  solution  again  until  the  color  remains  permanent  on  warming.  Then 
boil,  when  the  irritating  vapors  of  iodo-acetone  will  attack  the  eyes.  Ace- 
tone does  not  give  this  reaction. 

Detection  of  Acetone  and  Acetoacetic  Acid  in  the  Urine.  Before  testing 
for  acetone  test  for  acetoacetic  acid;  as  this  acid  gradually  decomposes  on 
allo\\ing  the  urine  to  stand,  the  specimen  must  be  as  fresh  as  possible.  In 
the  presence  of  acetoacetic  acid  the  urine  gives  the  above-mentioned  tests. 
In  testing  for  acetone  in  the  presence  of  acetoacetic  acid  make  the  urine 
sUghtly  allcaUne  and  shake  in  a  separator)^  funnol  with  ether  free  from 
alcohol  and  acetone.  Remove  the  ether  and  shake  it  with  water,  which 
takes  up  the  acetone,  and  test  for  acetone  in  the  watery  solution. 

In  the  absence  of  acetoacetic  acid  the  acetone  may  be  tested  for  directly 
in  the  urine;  this  may  be  done  by  Penzoldt's  test.  This  test,  which  is 
only  approximate,  is  of  value  only  when  the  urine  contains  a  considerable 
amount  of  acetone.  For  a  more  accurate  test  we  distill  at  least  250  c.c. 
of  the  urine  faintly  acidified  with  sulphuric  acid,  care  being  taken  to  have 
a  good  condensation.  ]\lost  of  the  acetone  is  contained  in  the  first  10-20 
c.c.  of  the  distillate.  A  better  result  may  be  obtained  by  distilling  a  large 
quantity  of  urine  until  about  ro  has  been  distilled  off,  acidify  the  distillate 
with  hydrochloric  acid,  redistill  and  repeat  this  several  times,  collecting 
the  first  portion  of  each  distillation.  The  final  distillate  is  used  for  the 
above  reactions. ^  Salkowski  and  Borchardt  have  called  attention  to 
the  fact  that  in  the  distillation  of  an  acidified  urine  containing  sugar  for 
the  detection  or  estimation  of  acetone  a  substance  giving  iodoform  can  be 
formed  from  the  sugar  if  the  distillation  is  carried  too  far.  According  to 
Borchardt  2  the  urine  must  therefore  first  be  diluted  with  water  or  the 
concentration  prevented  by  the  addition  of  water  dropwise  during  distil- 
lation. 

The  quantitative  estimation  of  acetone  in  the  urine  is  done  by  convorting 
it  first  into  iodoform.  The  urine  is  acidified  with  acetic  acid  (according  to 
HupPERT,  1-2  c.c.  50  per  cent  acetic  acid  for  ever\^  100  c.c.  urine)  and 
distilled.  The  quantity  of  acetone  in  the  distillate  is  best  determined 
according  to  ^Iessinger  and  Huppert's  method  by  determining  volu- 
me trically  the  quantity  of  iodine  used  in  the  formation  of  iodoform.  In 
regard  to  this  method  and  its  execution  the  reader  is  referred  to  Huppert- 
Neubauer.^ 

CH3 

/3-Oxybutyric  Acid,  C4H803  =  CHOH.    The  occurence  of   this  acid  in 

CH2 
COOH 
the  urine  w^as  first  positively  shown  by  ]\Iinkowski,  KiJLZ,   and  Stadel- 
MANN.4     It  occurs  especially  in  severe  cases  of  diabetes,  when  it  may  form 

*See  also  Salkowski,  Pfliiger's  Arch.,  56. 

*  Hofmeister's  Beitrage,  8. 

'  Hamanalyse,  760,  and  also  Geelmuyden,  Zeitschr.  f.  anal.  Chem.,  35,  and  Vaubel, 
Chem.  Centralbl.,  1905,  1,  1617. 

*  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  IS  and  19;  Stadelmann,  ibid.,  17; 
KiUz,  Zeitschr.  f.  Biologie,  20  and  23. 


674  URINE. 

the  largest  portion  of  the  acetone  bodies  (Magnus-Levy,  Geelmuyden)  .  It 
has  also  been  observed  in  scarlet  fever,  measles,  in  scurvy,  and  in  diseases 
of  the  brain  with  abstinence.  It  seems  to  be  always  accompanied  with 
acetoacetic  acid. 

The  /3-oxy butyric  acid  ordinarily  forms  an  odorless  syrup,  but  may  also 
be  obtained  as  crystals.  It  is  readily  soluble  in  water,  alcohol,  and  ether. 
It  is  levorotatory ;  (a)u=  —24.12°  for  solutions  of  1-11  per  cent  and  has 
a  disturbing  action  upon  the  determination  of  sugar  by  means  of  the  polari- 
scope.  It  is  not  precipitated  by  basic  lead  acetate  or  by  ammoniacal  lead 
acetate,  neither  does  it  ferment.  On  boiling  with  water,  especially  in  the 
presence  of  a  mineral  acid,  this  acid  decomposes  into  a-crotonic  acid, 
which  melts  at  71-72°  C,  and  water:  CH3.CH(OH).CH2.COOH  =  H20  + 
CHs.CH-.CH.COOH.  It  yields  acetone  on  oxidation  with  a  chromic-acid 
mixture. 

Detection  of  ^-Oxyhutyric  Acid  in  the  Urine.  If  a  urine  is  still  levo- 
gyrate  after  fermentation  with  yeast,  the  presence  of  oxy butyric  acid  is 
probable.  A  further  test  may  be  made,  according  to  Kulz,  by  evaporating 
the  fermented  urine  to  a  symp  and,  after  the  addition  of  an  equal  volume 
of  concentrated  sulphuric  acid,  distilling  directly  without  cooling,  a-cro- 
tonic acid  is  produced  which  distills  over,  and,  after  collecting  in  a  test- 
tube,  crystals  which  melt  at  -|-72°  C.  separate  on  cooUng.  If  no  crystals 
are  obtained,  shake  the  distillate  with  ether,  evaporate,  and  test  the  melt- 
ing-point of  the  residue  which  has  been  washed  with  water.  According 
to  Minkowski  the  acid  may  be  isolated  as  a  silver  salt.^ 

The  Quantitative  Estimation  may  be  performed  as  follow^s,  according 
to  Bergell:^  100-300  c.c.  of  the  sugar-free  urine  or  fermented  urine 
is  made  slightly  alkaline  with  sodium  carbonate  and  concentrated  to  a 
syrup.  This,  on  cooling,  is  rubbed  with  synipy  phosphoric  acid  (keeping 
it  cool),  anhydrous  copper  sulphate  (20-30  grams),  and  fine  sand,  and  the 
dry  mass  thoroughly  extracted  with  anhydrous  ether  in  an  extraction 
apparatus.  The  residue  after  the  evaporation  of  the  ether  is  dissolved 
in  water  and  decolorized,  if  necessary,  with  animal  charcoal,  and  the  quan- 
tity of  the  acid  calculated  from  the  polarization.  Other  methods  have  been 
suggested  by  Darmstadter,  Boekelman  and  Bouma,  and  Magnus-Levy.^ 

Ehrlich's  *  Urine  Test.  Mix  250  c.c.  of  a  solution  which  contains  50  c.c. 
HCl  and  1  gram  of  sulphaiiilic  acid  in  one  liter  with  5  c.c.  of  a  §  per  cent  solution 
of  sodium  nitrite  (which  produces  very  little  of  the  active  body,  sulphodiazo- 
benzene).  In  performing  this  test  treat  the  urine  with  an  equal  volume  of  this 
mixture  and  then  supersaturate  with  ammonia.  Normal  urine  will  become 
yellow  thereby,  or  orange  after  the  addition  of  ammonia  (aromatic  oxyacids  may 

'  Arch.  f.  exp.  Path.  ii.  Pharm.,    18,  35;  Zeitschr.  f.  anal.  Cliem.,  24,  153. 

2  Zeitschr.  f.  physiol.  Chem.,  33. 

'  Darmstadter,  ibi/L,  37;  Boekelmann  and  Bouma,  see  Maly's  Jahresber.,  31; 
Magnus-Levy,  Arch  f.  exp.  Path  u.  Pharm.  45. 

*  Ehrlich,  Zeitschr.  f.  kUn.  Med..  5.  See  also  Clemens,  Deutsch.  Arch.  f.  klin. 
Med.,  63  (literature). 


CYSTINE.  675 

sometime,  after  a  certain  time  give  red  azo  bodies  which  color  the  upper  layer  of 
the  phosphate  sediment).  In  pathological  urines  there  sometime.:!  occur.-?  (and  this 
is  the  charactpristic  Hinzo  reaction)  a  primary  yellow  coloration,  with  a  very 
marked  secondary  r^^d  coloration  on  the  addition  of  ammonia,,  and  the  frotli  is 
also  tinged  with  red.  The  upper  layer  ot  ilie  sedlineat  udcom^s  giejni./a.  Tae 
body  which  gives  this  reaction  is  unknown,  but  it  occurs  especially  in  the  urine 
of  typhoid  patients  (Ehrlich).  Opinions  differ  in  regard  to  the  significance  of 
this  reaction.  The  fact  that  the  antoxyproteic  acid  gives  this  reaction  as  above 
stated  (page  612)  is  of  interest. 

Another  urine  test  suggested  by  Ehrlich  consists  in  adding  a  hydrochloric 
acid  containing  2  per  cent  dimethylaminobenzaldehyde  to  the  urine;  normal  urines 
are  colored  faintly  red,  while  certain  pathological  urines  become  cherry-red. 
The  cause  of  this  reaction  is  not  sufficiently  known;  according  to  Neubaubr  '  it 
appears  to  be  connected  with  the  urobilinogen. 

Rosenbach's  urine  test,  which  consists  in  adding  nitric  acid  drop  by  drop 
to  the  boiling-hot  urine  and  obtaining  a  claret-red  coloration  and  a  bluish-red 
foam  on  shaking,  depends  upon  the  formation  of  indigo  substances,  especially 
indigo  red.^ 

Fat  in  the  Urine.  The  elimination  of  a  urine  which  in  appearance  and  rich- 
ness in  fat  resembles  chyle  is  called  chyluria.  It  habitually  contains  a  proteid  and 
often  fibrin.  Chyluria  occurs  mostly  in  the  inhabitants  of  the  tropics.  Lipuria, 
or  the  elimination  of  fat  with  the  urine,  may  appear  in  apparently  healthy  persons, 
sometimes  with  and  sometimes  without  albuminuria,  in  pregnancy,  and  also  in 
certain  diseases,  as  in  diabetes,  poi.soning  \vith  phosphorus,  and  fatty  degeneration 
of  the  kidneys. 

Fat  is  usually  detected  by  the  microscope.  It  may  also  be  dissolved  with 
ether,  and  may  invariably  be  detected  by  evaporating  the  urine  to  dryness  and 
extracting  the  residue  with  ether. 

Cholesterin  is  also  sometimes  found  in  the  urine  in  chyluria  and  in  a  few  other 
cases. 

Amino-acids.  Leucine  and  tyrosine  have  been  repeatedly  found  by 
the  older  methods  in  urine,  especially  in  acute  yello^v  atrophy  of  the  liver, 
in  acute  phosphoiiis-poisoning,  and  in  severe  cases  of  typhoid  and  smallpox. 
Since  the  use  of  /^-naphthalene  sulphochloride  has  been  used  in  the  detection 
of  amino-acids  these  bodies  have  not  only  been  repeatedly  found  in  normal 
urine  (glycocoU,  see  page  614.)  but  also  in  pathological  urines.  Besides  an 
increased  amount  of  glycocoU  in  certain  cases  of  gout  (Alex.  Ignatowski) 
and  the  finding  of  tyrosine  and  leucine  in  cystinuria  (Abderhalden  and 
Schittenhelm)  and  in  certain  other  cases,  Abderhaldex  and  Barker  ^ 
have  also  found  phenylalanine  (besides  glycocoU,  tyrosine,  and  leucine)  in 
the  urine  in  dogs  after  phosphorus  poisoning. 

Cystine  (see  page  92).     Baumann  and  Goldmaxn*  claim  that  a  sub- 

>  See  Proscher,  Zeitschr.  f.  physiol.  Chem.,  31,  and  Clemens,  Deutsch.  Arch.  f. 
klin.  Med.,  71;  Neubauer,  Centralbl.  f.  Physiol  ID,  145. 

^  S.e  Rosin,  Virchow's  Arch.,  123. 

"  ^'gnatowski,  Zeitschr.  f.  physiol.  Chem.  42;  Abderhalden  and  Schitten'.ielm,  ibid. 
•15;  Abderhalden  and  Bark-cr,  ibid.  42. 

*  Baumann,  Zeitschr.  f,  physiol.  Cnem.'  8.  In  regard  to  the  literature  on  cystine 
see    Brenzinger,    tbid.,  16;    Baumann   and   Goldmann,    ibid.,  12;   Baumann  and  v. 


676  URINE. 

stance  similar  to  cystine  occurs  in  very  small  amounts  in  normal  urine. 
This  substance  occurs  in  large  quantities  in  the  urine  of  dogs  after  poison- 
ing with  phosphorus.     Cystine  itself  is  only  found  with  positiveness,  and 
even  then  ver\'  rarely,  in  urinaiy  calculi  and  in  pathological  urines,  irom 
which  it  may  separate  as  a  sediment.     Cystinuria  occurs  oftener  in  men 
than  in  women.     Baumann  and  v.  Udraxszky  found  in  urine  in  cystinuria 
the  two  diamines,  cadaverine  (pentamethylendiamine)  and  -putrescine  (tetra- 
methylendiamine),   wliich  are  produced  in  the  putrefaction  of  proteins. 
These  two  diamines  were  also  found  in  the  contents  of  the  intestine  in 
cystinuria,  while  under  normal  conditions  they  are  not  present.     Hammar- 
STEX  therefore   considers  that  perhaps  some  connection  exists  between 
the  formation  of  diamines  in  the  intestine,  by  the  peculiar  i3utrefaction 
in  cystinuria,  and  cystinuria  itself.     This  is  less  probable  and  cystinuria  is, 
as  generally  admitted,  rather  an  anomaly  in  the  protein  metabolism  where 
the  cystine  for  unknown  reasons  is  not  destroyed  as  ordinarily,  although 
sometimes  those  having  cystinuria  can  quantitatively  destroy  the  cystine 
introduced.     Cases  of  cystinuria  may  occur  with  or  without  the  occurrence 
of  diamines  in  the  urine,  and  only  rarely  are  the  diamines  found  in  the 
urine  as  well  as  in  the  feces,  which  perhaps  depends  upon  the  fact,  as  found 
bv  Cammridge  and  Garrod  ^  in  one  case,  that  the  diamines  occur  only 
from  time  to  time  in  the  feces.    The  properties  and  reactions  of  cystine 
have  been  given  on  pages  92  and  93. 

Cystine  is  easily  prepared  from  cystine  calculi  by  dissohdng  them  in 
alkali  carbonate,  precipitating  the  solution  with  acetic  acid,  and  redissolv- 
ing  the  precipitate  in  ammonia.  The  cystine  crystallizes  on  the  spontane- 
ous evaporation  of  the  ammonia.  The  cystine  dissolved  in  the  urine  is 
detected,  in  the  absence  of  proteid  and  sulphuretted  hydrogen,  by  boiling 
with  allvali  and  testing  with  a  lead  salt  or  sodium  nitropmsside.  To  isolate 
cystine  from  the  urine,  acidify  the  urine  strongly  with  acetic  acid.  The 
precipitate  containing  cystine  is  collected  after  twenty -four  hours  and 
dio-ested  with  hydrochloric  acid,  which  dissolves  the  cystine  and  calcium 
oxalate,  leaving  the  uric  acid  undissolved.  Filter,  supersaturate  the  filtrate 
with  ammonium  carbonate,  and  treat  the  precipitate  with  ammonia,  which 
dissolves  the  cystine  and  leaves  the  calcium  oxalate.  Filter  again  and  pre- 
cipitate with  acetic  acid.  The  precipitated  cystine  is  identified  by  the 
microscope  and  the  above-mentioned  reactions.  Cystine  as  a  sediment  is 
identified  by  the  microscope.  It  must  be  purified  b}'  dissoh-ing  in  ammonia 
and  precipitating  with  acetic  acid  and  then  further  tested.  Traces  of  dis- 
solved cN'stine  may  be  detected  by  the  production  of  benzoyl-cystine,  ac- 
cording to  Baumaxx  and  Goldman. 


Udranszky,  iibid.,  13;  Stadthagen  and  Brieger.  Berlin,  klin.  Wochenschr.,  1889;  Cam- 
midge  and  Ciarrod,  Journ  of  Path,  and  Bacteriol.  1900  (literature  on  diamines  in  thj 
urine  and  feces). 

'  Joum.  of  Path,  and  Bacteriol  .  YM  0. 


URINARY   SEDIMENTS  AND   CALCULI.  677 


VII.  Urinary  Sediments  and  Calculi. 

Urinan^  sediment  is  the  more  or  less  abundant  deposit  which  is  found 
in  the  urine  after  standing.  This  deposit  maj-  consist  partly  of  organized 
and  partly  of  non-organized  constituents.  The  first,  consisting  of  cells  of 
various  kinds,  yeast-fungi,  bacteria,  spermatozoa,  casts,  etc.,  must  be 
investigated  by  means  of  the  microscope,  and  the  following  only  applies 
to  the  non-organized  deposits. 

As  previously  mentioned  (page  543),  the  urine  of  healthy  indi\aduals 
may  sometimes,  even  on  voiding,  be  cloudy  on  account  of  the  phosphates 
present,  or  become  so  after  a  little  while  because  of  the  separation  of  urates. 
As  a  mle,  urine  just  voided  is  clear,  and  after  cooUng  shows  only  a  faint 
cloud  (nubecula)  which  consists  of  urine  mucoid,  a  few  epithelium-cells 
mucous  corpuscles,  and  urate  particles.  If  an  acid  urine  is  allowed  to  stand, 
it  will  gradually  change;  it  becomes  darker  and  deposits  a  sediment  eon- 
sisting  of  uric  acid  or  urates,  and  sometimes  also  calcium-oxalate  crj'stals, 
in  which  yeast-fungi  and  bacteria  are  often  to  be  seen.  This  change,  which 
the  earlier  investigators  called  "acid  fermentation  of  the  urine,"  is 
generally  considered  as  an  exchange  of  the  dihydrogen  alkali  phosphates 
with  the  urates  of  the  urine.  ^lonohydrogen  phosphates  besides  acid  urates 
or  free  uric  acid  or  a  mixture  of  both,  according  to  conditions,^  are  hereby 
formed. 

Sooner  or  later,  sometimes  only  after  several  weeks,  the  reaction  of  the 
original  acid  urine  changes  and  becomes  neutral  or  alkaline.  The  urine  has 
now  passed  into  the  "alic^line  fermentation,"  which  consists  in  the 
decomposition  of  the  urea  into  carbon  dioxide  and  ammonia  b}-  means  of 
lower  organisms,  micrococcus  urese,  bacterium  ureae,  and  other  bacteria. 
MuscuLUs  2  has  isolated  an  enzyme  from  the  micrococcus  urea?  which 
decomposes  urea,  is  soluble  in  water  and  is  called  urease.  During  the 
alkaline  fermentation  volatile  fatty  acids,  especially  acetic  acid,  may  be 
produced,  chiefly  by  the  fermentation  of  the  carbohydrates  of  the  urine 
(Salkowski^).  A  fermentation  by  which  nitric  acid  is  reduced  to  nitrous 
acid,  and  another  where  sulphuretted  hydrogen  is  produced,  may  sometimes 
occur. 

When  the  alkaline  fermentation  has  advanced  only  so  far  as  to  render 
the  reaction  neutral,  there  often  occur  in  the  sediment  fragments  of  uric- 
acid  crA'stals,  sometimes  covered  with  prismatic  cr>'stals  of  alkali  urate; 
dark -colored  spheres  of  ammonium  urate,  cr\-stals  of  calcium  oxalate,  and 


'  See  Huppert-Neubauer  10.  Aufl.,  and  A.  Ritter,  Zeitschr.  f.  Biologie,  35. 

^  Musculus,  Pfluger's  Arch  ,  12. 

^  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  13. 


678  URINE. 

sometimes  cn'stallized  calcium  phosphate  are  also  found.  Cn-stals  of 
ammonium-magnesium  phosphate  (triple  phosphate)  and  spherical  ammo- 
uium  urate  are  specially  characteristic  of  alkahne  fermentation.  The  urine 
in  alkaline  fermentation  becomes  paler  and  is  often  covered  with  a  fine 
membrane  which  contains  amorphous  calcium  phosphate  and  glistening 
crystals  of  triple  phosphate  and  numerous  micro-organisms. 


Won-organized  Sediments. 

Uric  Acid.  This  acid  occurs  in  acid  urines  as  colored  crj^stals  wliich  are 
identified  partly  by  theii'  form  and  partly  by  their  property  of  gi\ing  the 
murexid  test.  On  warming  the  urine  they  are  not  dissolved.  On  the 
addition  of  caustic  alkali  to  the  sediment  the  cr\'stals  dissolve,  and  when  a 
drop  of  this  solution  is  placed  on  a  microscope-slide  and  treated  ^x\ih.  a  drop 
of  hvdrochloric  acid  small  crj-stals  of  uric  acid  are  obtained  which  can  be 
easily  seen  under  the  microscope. 

Acid  Urates.  These  occur  only  in  the  sediment  of  acid  or  neutral 
urines.  They  are  amorphous,  clay -yellow,  brick-red,  rose-colored,  or 
brownish  red.  They  differ  from  other  sediments  in  that  they  dissolve  on 
warming  the  urine.  They  give  the  murexid  test,  and  small  microscopic 
cr}'stals  of  uric  acid  separate  on  the  addition  of  hydrochloric  acid.  Crys- 
talline alkali  urates  occur  vers-  rarely  in  the  urine,  and  as  a  rule  only  in 
such  as  have  tecome  neutral  but  not  alkaline  by  alkaline  fermentation. 
The  cr\'stals  are  somewhat  similar  to  those  of  neutral  calcium  phosphate; 
they  are  not  dissolved  by  acetic  acid,  however,  but  give  a  cloudiness  there- 
with due  to  small  cr}'stals  of  uric  acid. 

Ammonium  urate  may  indeed  occur  as  a  sediment  in  a  neutral  urine 
which  at  first  w^as  strongly  acid  and  has  become  neutralized  by  the  alkaline 
fermentation,  but  it  is  only  characteristic  of  ammoniacal  urines.  This 
sediment  consists  of  yellow  or  brownish  rounded  spheres  which  are  often 
covered  with  thorny -shaped  prisms  and,  because  of  this,  are  rather  large 
and  resemble  the  thorn-apple.  It  reacts  to  the  murexid  test.  It  is  dis- 
solved by  alkalies  with  the  development  of  ammonia,  and  crj-stals  of  uric 
acid  separate  on  the  addition  of  hydrochloric  acid  to  this  solution. 

Calcium  oxalate  occurs  in  the  sediment  generally  as  small,  shining, 
strongly  refractive  quadratic  octahedra,  which  on  microscopical  examina- 
tion remind  one  of  a  letter-envelope.  The  cr}-stals  can  only  be  mistaken 
for  small,  not  fully  developed  cr}'stals  of  ammonium-magnesium  phos- 
phate. The}'^  differ  from  these  by  their  insolubility  in  acetic  acid.  The 
oxalate  may  also  occur  as  flat.  oval,  or  nearly  circular  disks  \\-ith  central 
ca\nties  which  from  the  side  apj^ear  like  an  hour-glass.  Calcium  oxalate 
mav  occur  as  a  sediment  in  an  acid  as  well  as  in  a  neutral  or  alkaline  urine. 


NON-ORGANIZED    SEDLMENTS.  679 

The  quantity  of  calcium  oxalate  separated  from  the  urine  as  sediment 
depends  not  only  upon  the  amount  of  this  salt  present  but  also  upon  the 
acidity  of  the  urine.  The  solvent  for  the  oxalate  in  the  urine  seems  to  ]ye 
the  diacid  alkah  phosphate,  and  the  greater  the  quantity  of  this  salt  in  the 
urine  the  greater  the  quantity  of  oxalate  in  solution,  ^^^len.  as  pre\'iously 
mentioned  (page  677) ,  the  simple-acid  phosphate  is  formed  from  the  diacid 
phosphate,  on  allowing  the  urine  to  stand,  a  corresponding  part  of  the  oxa- 
late may  be  separated  as  sediment. 

Calcium  carbonate  occurs  in  considerable  quantities  as  sediment  in  the 
urine  of  herbivora.  It  occurs  in  but  small  quantities  as  a  sediment  in 
human  urine,  and  in  fact  only  i;i  alkaline  urines.  It  either  has  almost  the 
same  appearance  as  amorphous  calcium  oxalate  or  it  occurs  as  somewhat 
larger  spheres  with  concentric  bands.  It  dissolves  in  acetic  acid  with  the 
generation  of  gas,  which  differentiates  it  from  calcium  oxalate.  It  is  not 
yellow  or  brown  like  ammonium  urate,  and  does  not  give  the  murexid  test. 

Calcium  Phosphate.  The  c.\lcium  triphosphate,  Ca3(P04)2^  which 
occurs  only  in  alkaUne  urines,  is  always  amorphous  and  occurs  partly  as  a 
colorless,  ver}-  fine  powder  and  partly  as  a  membrane  coiLsisting  of  ven,' 
fine  granules.  It  differs  from  the  amorphous  urates  in  that  it  is  colorless, 
dissolves  in  acetic  acid,  but  remains  undissolved  on  warming  the  urine. 
Calcium  diphosphate.  CaHP04-L2H20.  occurs  in  neutral  or  only  in  vers- 
faintly  acid  urine.  It  is  found  sometimes  as  a  thin  film  covering  the  urine 
and  sometimes  as  a  Sediment.  In  crj'stallizing.  the  crj-stals  may  be  single, 
or  they  may  cross  one  another,  or  they  may  be  arranged  in  groups  of  color- 
less, wedge-shaped  cr^-stals  whose  wide  end  is  sharply  defined.  These  cr^-s- 
tals  differ  from  crj^stalline  alkali  urates  in  that  they  dissolve  without  a 
residue  in  dilute  acids  and  do  not  give  the  murexid  test 

Calcium  sulphate  occurs  very  rarely  as  a  sediment  in  strongly  acid  urine.  It 
appears  as  long,  thin,  colorless  needles,  or  generally  as  plates  grouped  together. 

Ammonium-magnesium  phosphate,  triple  phosphate,  mav  separate 
from  an  amphoteric  urine  in  the  presence  of  a  sufficient  quantity  of  am- 
monium salts,  but  it  is  generally  characteristic  of  a  urine  wliich  is  ammo- 
niacal  through  alkaline  fermentation.  The  ciystals  are  so  large  that  they 
may  be  seen  with  the  unaided  eye  as  colorless  glistening  particles  in  the 
sediment,  on  the  walls  of  the  vessel,  and  in  the  film  on  the  surface  of  the 
urine.  This  salt  forms  large  prismatic  cr^-stals  of  the  rhombic  svstem 
(coffin-shaped)  which  are  easily  soluble  in  acetic  acid.  Amorphous  magne- 
sium, triphosphate,  ^Ig3(P04)2,  occurs  with  calcium  triphosphate  in  urines 
rendered  alkaline  by  a  fixed  alkali.  CiystalUne  magnesium  phosphate, 
]\Ig3(P04)2+22H20,  has  been  ob.served  in  a  few  cases  in  human  urine  (also 
in  horse's  urine)  as  strongly  refracti\-e.  long  rhombic  plates. 


68C  URINE. 

Kyestein  is  the  film  which  appears  after  a  little  while  on  the  surface  of  the  urine. 
This  coating,  which  was  foruierly  considered  as  characteristic  of  urine  in  preg- 
nancy, contains  various  elements,  such  as  fungi,  vibriones,  epithelium-cells,  etc. 
It  often  contains  earthy  phosphates  and  triple-phosphate  crystals. 

As  more  rare  sediments  we  find  cystine,  tyrosine,  hippuric  acid,  xanthine,  hoema- 
toidine.  In  alkaline  urine  blue  crystals  of  indigo  may  also  occur,  due  to  a  decom- 
position of  indoxyl-glucuronic  acid. 


Urinary  Calculi. 

Besides  certain  pathological  constituents  of  the  urine,  all  those  urinary 
constituents  which  occur  as  sediments  take  part  in  the  formation  of  urinary 
calculi.  Ebstein  ^  considers  the  essential  difference  between  an  amorphous 
or  crystalUne  sediment  in  the  urine  on  one  side  and  urinary  sand  or  large 
calculi  on  the  other  to  be  the  occurrence  of  an  organic  frame  in  the  latter. 
As  the  sediments  which  appear  in  normal  acid  urine  and  in  a  urine  alkaline 
through  fermentation  are  diverse,  so  also  are  the  urinary  calculi  which 
appear  under  corresponding  conditions. 

If  the  formation  of  a  calculus  and  its  further  development  take  place 
in  an  undecomposed  urine,  it  is  called  a  primary  formation.  If,  on  the  con- 
trary, the  urine  has  undergone  alkaline  fermentation  and  the  ammonia 
formed  thereby  has  given  rise  to  a  calculus  formation  by  precipitating 
ammonium  urate,  triple  phosphate,  and  earthy  phosphates,  then  it  is  called 
a  SECONDARY  formation.  Such  a  formation  takes  place,  for  instance,  when 
a  foreign  body  in  the  bladder  23roduces  catarrh  accompanied  by  alkaline 
fermentation. 

We  discriminate  between  the  nucleus  or  nuclei — if  such  can  be  seen — 
and  the  different  layers  of  the  calculus.  The  nucleus  may  be  essentially 
different  in  different  cases,  for  quite  frequently  it  consists  of  a  foreign  body 
introduced  into  the  bladder.  The  calculus  may  have  more  than  one  nu- 
cleus. In  a  tabulation  made  l)y  Ultzmann  of  545  cases  of  vesicular  calcuh, 
the  nucleus  in  80.9  per  cent  of  the  cases  consisted  of  uric  acid  (and  urates); 
in  5.6  per  cent,  of  calcium  oxalate;  in  8.6  per  cent,  of  earthy  phosphates; 
in  1.4  per  cent,  of  cystine;  and  in  3.5  per  cent,  of  some  foreign  body. 

During  the  growth  of  a  calculus  it  often  happens  that,  for  some  reason 
or  other,  the  original  calculus-forming  substance  is  covered  with  another 
layer  of  a  different  substance.  A  new  layer  of  the  original  substance  may 
deposit  on  the  outside  of  this,  and  this  process  may  be  repeated.  In  this 
way  a  calculus  consisting  originall}^  of  a  simple  stone  may  be  converted  into 
a  so-called  compound  stone  with  several  layers  of  different  substances. 
Such  calculi  are  always  formed  when  a  primary  is  changed  into  a  secondary 
formation.     By  the  continued  action  of  an  alkaline  urine  containing  pus, 

'  Die  Natur  und  Behandlung  der  Harnsteine.     Wiesbaden,  1884. 


URINARY  CALCULI.  681 

the  primary  constituents  of  an  originally  primary  calculus  may  be  partly 
dissolved  and  be  replaced  by  phosphates.  .Metamorphosed  urinary  calculi 
are  formed  in  this  way. 

Uric-acid  calculi  are  very  abundant.  They  are  variable  in  size  and 
form.  The  size  of  the  bladder-stone  varies  from  that  of  a  pea  or  bean  to 
that  of  a  goose-egg.  Uric-acid  stones  are  always  colored :  generally  they 
are  grayish  yellow,  yellowish  brown,  or  pale  red-brown.  The  upper  surface 
is  sometimes  entirely  even  or  smooth,  sometimes  rough  or  uneven.  Next 
to  the  oxalate  calculus  the  uric-acid  calculus  is  the  hardest.  The  fractured 
surface  shows  regular  concentric,  unequally  colored  layers  which  may  often 
be  removed  as  shells.  These  calculi  are  formed  primarily.  Layers  of  uric 
acid  sometimes  alternate  with  otlier  layers  of  primary  formation,  most 
frequently  with  layers  of  calcium  oxalate.  The  simple  uric-acid  calculus 
leaves  very  little  residue  when  burnt  on  a  platinum  foil.  It  gives  the 
murexid  test,  but  there  is  no  material  development  of  ammonia  when  acted 
on  by  caustic  soda. 

Ammonium  urate  calculi  occur  as  primary  calculi  in  new-born  or  nursing 
infants,  rarely  in  grown  persons.  They  often  occur  as  a  secondary  forma- 
tion. The  primary  stones  are  small,  with  a  pale-yellow  or  dark-yellowish 
surface.  When  moist  they  are  almost  like  dough;  in  the  dry  state  they 
are  earthy,  easily  crumbling  into  a  pale  powder.  They  give  the  murexid 
test  and  develop  much  ammonia  with  caustic  soda. 

Calcium-oxalate  calculi  are,  next  to  uric-acid  calculi,  the  most  abundant. 
They  are  either  smooth  and  small  (hemp-seed  calculi)  or  larger,  of  the 
size  of  a  hen's  egg,  with  rough,  uneven  surface,  or  their  surface  is  covered 
with  prongs  (mulberry  calculi).  These  calculi  produce  bleeding  easily, 
and  therefore  they  often  have  a  dark-brown  surface  due  to  decomposed 
blood-coloring  matters.  Among  the  calculi  occurring  in  man  these  are  the 
hardest.  They  dissolve  in  hydrochloric  acid  without  developing  gas,  but 
are  not  soluble  in  acetic  acid.  After  gently  heating  the  powder,  it  dissolves 
in  acetic  acid  with  frothing.  With  more  intense  heat  it  becomes  alkaline, 
due  to  the  production  of  quicklime. 

Phosphate  Calculi.  These,  which  consist  mainly  of  a  mixture  of  the 
normal  phosphate  of  the  alkaline  earths  with  triple  phosphate,  may  be 
very  large.  They  are  as  a  rule  of  secondary  formation  and  contain  besides 
these  phosphates  also  some  ammonium  urate  and  calcium  oxalate.  These 
calculi  ordinarily  consist  of  a  mixture  of  three  constituents  —  earthy 
phosphate,  triple  phosphate,  and  ammonium  urate  —  surrounding  a 
foreign  body  as  a  nucleus.  Their  color  is  variable  —  white,  dingy  white, 
pale  yellow,  sometimes  violet  or  lilac-colored  (from  indigo  red).  The 
surface  is  always  rough.  Calculi  consisting  of  triple  phosphate  alone  are 
seldom   found.     They   are   ordinarily   small,   with   granular  or   radiated 


682  URINE. 

crystalline  fracture.  Stones  of  mono-acid  calcium  phosphate  are  also 
seldom  obtained.  They  are  white  and  have  beautiful  crystalline  texture. 
The  phosphatic  calculi  do  not  burn  up,  the  powder  dissolves  in  acid  with- 
out effervescence,  and  the  solution  gives  the  reactions  for  phosphoric  acid 
and  the  alkaline  earths.  The  triple-phosphate  calculi  generate  ammonia 
on  the  addition  of  an  alkali. 

Calduyn-carhonate  calculi  occur  chiefly  in  herbivora.  The}^  are  seldom  found 
in  man.  They  have  mostly  chalky  properties,  and  are  ordinarily  white.  They 
are  completely  or  in  great  part  dissolved  by  acids  with  effervescence. 

Cystine  calculi  occur  but  seldom.  They  are  of  primary  formation,  of  various 
sizes,  sometimes  as  large  as  a  hen's  egg.  They  have  a  smooth  or  rough  surface, 
are  white  or  pale  yellow,  and  have  a  crystalline  fracture.  They  are  not  very 
hard  and  are  consumed  almost  entirel}^  on  the  platinum  foil  burning  with  a  bluish 
flame.     They  give  the  above-mentioned  reactions  for  cystine. 

Xanthine  calculi  are  very  rarely  found.  They  are  also  of  primary  formation. 
They  vary  from  the  size  of  a  pea  to  that  of  a  hen's  egg.  They  are  whitish,  yel- 
lowish-brown or  cinnamon-brown  in  color,  of  medium  hardness,  with  amorphous 
fracture,  and  on  rubbing  appear  like  wax.  They  burn  up  completely  when 
heated  on  a  platinum  foil.  They  give  the  xanthine  reaction  with  nitric  acid  and 
alkali  but  this  must  not  be  mistaken  for  the  murexid  test. 

Urostealith  calculi  have  been  ob.served  only  a  few  times.  In  the  moist  state 
they  are  soft  and  elastic  at  the  temperature  of  the  body,  but  in  the  dry  state  they 
are  brittle,  with  an  amorphous  fracture  and  waxy  appearance.  They  burn  with 
a  luminous  flame  when  heated  on  platinum  foil  and  generate  an  odor  similar  to 
resin  or  shellac.  Such  a  calculus,  inve.stigated  by  Krl'kenberg,^  consisted  of 
paraffine  derived  from  a  paraffine  bougie  used  as  a  sound  on  the  patient.  Perhaps 
the  urostealith  calculi  observed  in  other  cases  had  a  similar  origin,  although  the 
substances  of  which  they  consisted  have  not  been  closely  studied.  Horbaczew- 
SKi  has  recently  analyzed  a  case  of  urostealith  which,  to  all  appearances,  was 
formed  in  the  bladder.  This  calculus  contained  25  p.  m.  water,  8  p.  m.  inorganic 
bodies,  117  p.  m.  bodies  insoluble  in  ether,  and  850  p.  m.  organic  bodies  soluble 
in  ether,  among  which  were  515  p.  m.  free  fatty  acids,  335  p.  m.  fat,  and  traces  of 
cholesterin.  The  fatty  acids  consisted  of  a  mixture  of  stearic,  palmitic,  and 
probably  myristic  acids. 

HoRB.\czEWSKi  ^  has  also  analyzed  a  bladder  stone  which  contained  958.7  p.  m. 
choledcrin. 

Fibrin  calculi  sometimes  occur.  They  consist  of  more  or  less  changed  fibrin 
coagulum.     On  burning  they  develop  an  odor  of  burnt  horn. 

The  chemical  investigation  of  urinary  calculi  is  of  great  practical  impor- 
tance. To  make  such  an  examination  actually  instructive  it  is  necessary 
to  investigate  separately  the  different  layers  which  constitute  the  cal- 
culus. For  this  purpose  saw  the  calculus,  previously  wrapped  in  paper, 
with  a  fine  saw  so  that  the  nucleus  becomes  accessible.  Then  peel  off  the 
different  layers,  or,  if  the  stone  is  to  be  kept,  scrape  off  enough  of  the 
powder  from  each  layer  for  examination.  This  powder  is  then  tested  by 
heating  on  the  platinum  foil.     It  must  not  be  forgotten  that  a  calculus 

'  Chem.  Untersuch.  z.  wissensch.  Med.,  2.     Cited  from  Maly's  Jahresber.,  19,  422. 
^  Zeitschr.  f.  physiol.  Chem.,  18. 


URIXARY  CALCULI.  683 

is  never  entirely  burnt  up,  and  also  that  it  is  never  so  free  from  organic 
matter  that  on  heating  it  does  not  carbonize.  Do  not.  therefore,  lay  too 
great  stress  on  a  very  insignificant  imburnt  residue  or  on  a  very  small 
amount  of  organic  matter,  but  consider  the  calculus  in  the  former  case 
as  completely  burnt  and  in  the  latter  as  unaffected. 

When  the  powder  is  in  great  part  burnt  up,  but  a  significant  quantity 
of  unburnt  residue  remains,  then  the  powder  in  question  contains  as  a 
rule  urates  mixed  with  inorganic  bodies.  In  such  cases  remove  the  urate 
with  boiling  water  and  then  test  the  filtrate  for  uric  acid  and  the  suspected 
bases.  The  residue  is  then  tested  according  to  the  following  schema  of 
Heller,  which  is  well  adapted  to  the  investigation  of  urinary  calculi. 
In  regard  to  the  more  detailed  examination  the  reader  is  referred  to  special 
works  on  the  subject. 


684 


uraxE. 


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CHAPTER  XVI. 
THE  SKIN  AND  ITS  SECRETIONS. 

In  the  structure  of  the  skin  of  man  and  vertebrates  many  different 
kinds  of  substances  occur  which  have  already  been  considered,  such  as 
the  constituents  of  the  epidermal  formation,  the  connective  and  fatty 
tissues,  the  nerves,  muscles,  etc.  Among  these  the  different  horn  struc- 
tures, the  hair,  nails,  etc.,  whose  chief  constituent,  keratin,  has  been 
spoken  of  in  another  chapter  (Chapter  II),  are  of  special  interest. 

The  cells  of  the  horny  structure  show,  in  proportion  to  their  age,  a 
different  resistance  to  chemical  reagents,  especially  fixed  alkalies.  The 
younger  the  horn-cell  the  less  resistance  it  has  to  the  action  of  alkalies; 
with  advancing  age  the  resistance  becomes  greater,  and  the  cell-membranes 
of  many  horn-formations  are  nearly  insoluble  in  caustic  alkalies.  Keratin 
occurs  in  the  horn  structure  mixed  with  other  bodies,  from  which  it  is 
isolated  with  difficulty.  Among  these  bodies  the  mineral  constituents  in 
many  cases  occupy  a  prominent  place  because  of  their  quantity.  Hair 
leaves  on  burning  5-70  p.  m.  ash,  which  may  contain  in  1000  parts  230 
parts  alkali  sulphates,  140  parts  calcium  sulphate,  100  parts  iron  oxide, 
and  even  400  parts  silicic  acid.  Dark  hair  on  burning  seems  generally, 
although  not  always,  to  yield  more  iron  oxide  than  blond.  The  nails  are 
rich  in  calcium  phosphate,  and  the  feathers  rich  in  silicic  acid,  which 
Drechsel/  claims  exists  in  part  in  organic  combination  as  an  ester. 

According  to  Gautier  and  Bertrand  ^  arsenic  also  occurs  in  the  epider- 
mal formations.  The  arsenic  is,  according  to  Gautier,  of  importance  in 
the  formation  and  growth  of  the  same,  and  on  the  other  hand  these  struc- 
tures, hair,  nails,  and  epidermis-cells,  are  of  great  importance  for  the 
excretion  of  arsenic. 

The  skin  of  invertebrates  has  been  the  subject,  in  a  few  cases,  of  chemi- 
cal investigation,  and  in  these  animals  various  substances  have  been 
found,  of  which  a  few,  though  little  studied,  are  worth  discussing.  Among 
these  bodies  tunicin,  which  is  found  especially  in  the  mantle  of  the  tuni- 


>  Centralbl.  f.  Physiol.,  11,  361. 

''Gautier,  Compt.  rend.,  129,  130,  131;  Bertrand,  ibid.,  134. 

685 


686  THE  SKIN  AND  ITS  SECRETIONS. 

cata,  and  the    widely  diffused  chitin,  found  in  the  cuticle-formation  of 
invertebrates,  are  of  interest. 

Tunicin.  Celluiose  seems,  according  to  the  investigations  of  Ambronn,  to 
occur  rather  extensively  in  the  animal  kingdom  in  the  arthropoda  and  the  mol- 
lusks.  It  has  been  known  for  a  long  time  as  the  mantle  of  the  tunicata,  and  this 
animal  cellulose  was  called  tunicin  by  Berthelot.  According  to  the  investiga- 
tions of  WiNTERSTEiN  there  does  not  seem  to  exist  any  marked  difference  between 
tunicin  and  ordinary  vegetable  cellulose.  On  boiling  with  dilute  acid  tunicin 
yields  dextrose,  as  shown  first  by  Franchimont  '  and  later  confirmed  by  Win- 

TERSTEIN. 

Chitin  is  not  found  in  vertebrates.  In  invertebrates  chitin  is  alleged 
to  occur  in  several  classes  of  animals;  but  it  can  be  positively  asserted 
that  true,  typical  chitin  is  found  only  in  articulated  animals,  in  which  it 
forms  the  chief  organic  constituent  of  the  shell,  etc.  According  to  Kraw- 
Kow^  chitin  of  the  shell,  etc.,  does  not  seem  to  occur  free,  but  in  com- 
bination with  another  substance,  probably  a  proteid-like  body.  Chitin 
also  occurs,  according  to  Gilsox  and  Wintersteix,^  in  certain  fungi. 

According  to  Suxdvik  the  formula  of  chitin  is  probably  C5oHjooN8038  + 
nCHfi),  where  n  may  vary  between  1  and  4.  According  to  Araki  it  has 
on  the  contrary  the  composition  CigHgoNjOj,.  According  to  Krawkow  the 
chit  ins  of  different  origin  show  different  behavior  with  iodine,  and  he 
therefore  concludes  that  there  must  exist  quite  a  group  of  chitins,  w^hich 
seem  to  be  amine  derivatives  of  different  carbohydrates,  such  as  dextrose, 
glycogen,  dextrins,  etc.  According  to  Zaxder  ^  only  two  chitins  exist, 
one  of  which  turns  violet  with  iodine  and  zinc  chloride,  and  the  other 
brown. 

Chitin  is  decomposed  on  boiling  with  mineral  acids  and  yields,  as 
shown  by  Ledderhose,  glucosamine  and  acetic  acid.  Schmiedeberg, 
therefore,  considers  chitin  as  a  probable  acetyl  acetic-acid  combination 
of  glucosamine.  Fraxkel  and  Kelly,^  on  the  contrarj^,  consider  chitin 
as  of  a  more  complicated  composition.  The  most  characteristic  cleavage 
product  obtained  by  them  was  a  chitosamine  acetylized  at  the  nitrogen 
atom,  CgHjLsOsN.COCHg,  and  a  second  product,  acetyldichitosamine, 
Cj4H2eOioN2,  which,  according  to  Araki,  has  the  same  composition  as 
chitosan  (see  below),  but  is  essentially  different  in  many  regards. 

^  Ambronn,  Maly's  Jahresber.,  20;  Berthelot,  Annal.  de  Chim,  et  Phys.,  o6,  Compt. 
rend.,  47;  AVinterstein,  Zeitschr.  f.  physiol.  Chem.,  18;  Franchimont,  Ber.  d.  deutsch 
chem.  Gesellsch.,  12. 

2  Zeitschr.  f.  Biologie,  29. 

'  Gilson,  Compt.  rend.,  120;  Winterstein,  Ber.  d.  deutsch.  chem.  GeseUsch.,  27  and 
28. 

^Sundvik,  Zeitschr.  f.  physiol.  Chem.,  5;  Araki.  ibid.  20;  Zander,  Pfliiger's  Arch.,  66. 

^  Ledderhose,  Zeitschr.  f.  physiol.  Chem.,  2  and  4;  Schmiedeberg,  Arch.  f.  exp.  Path, 
u.  Pharm.,  28;  Friinkel  and  Kelly,  Monatshefte  f.  Chem.,  23. 


CHITIX   AND   HYALIX.  687 

According  to  Hoppe-Seyler  and  Araki/  on  heating  chitin  with 
alkali  and  a  little  water  to  180°  C.  a  cleavage  takes  place  with  the  splitting 
off  of  acetic  acid,  and  the  formation  of  a  new  substance,  chitosan,  whose 
formula  according  to  Araki  is  C\4H.gN20io,  but  according  to  v.  Furth 
and  Russo^  more  Ukeiy  a  multiple  of  Ci3H2„X2Qij.  On  iieating  with  acetic 
anhydride  chitosan  is  converted  into  a  chitin-hke  substance  which  is  not 
identical  with  chitin.  Chitosan  is  insoluble  in  water  and  alkali,  but  dis- 
solves in  dilute  acids.  It  splits  into  acetic  acid  and  glucosamine  by  the 
action  of  hydrochloric  acid.  According  to  v.  Furth  and  Russo  on  acid 
cleavage  it  yields  25  per  cent  acetic  acid  and  60  per  cent  glucosamine. 
One  nitrogen  atom  corresponds  closely  to  1  molecule  acetic  acid  and  f 
molecule  glucosamine.  All  the  glucosamine  complexes  present  in  the 
chitosan  molecule  seem  to  be  acetylized.  According  to  Krawkow  the 
various  chitins  behave  differently  with  iodine  or  with  sulphuric  acid  and 
iodine,  in  that  some  are  colored  reddish  brown,  blue,  or  violet,  while 
others  are  not  colored  at  all. 

In  a  dry  state  chitin  forms  a  white,  brittle  mass  retaining  the  form  of 
the  original  tissue.  It  is  insoluble  in  boiling  water,  alcohol,  ether,  acetic 
acid,  dilute  mineral  acids,  and  dilute  alkalies. _  It  is  soluble  in  concen- 
trated acids.  It  is  dissolved  without  decomposing  in  cold  concentrated 
hydrochloric  acid,  but  is  decomposed  by  boiling  hydrochloric  acid.  When 
chitin  is  dissolved  in  concentrated  sulphuric  acid  and  the  solution  dropped 
into  boiling  water  and  then  boiled,  a  substance  is  obtained  (glucosamine, 
chitosamine)  which  reduces  copper  suboxide  in  alkaline  solutions. 

Chitin  may  be  easily  prepared  from  the  wings  of  insects  or  from  the 
shells  of  the  lobster  or  the  crab,  the  last-mentioned  having  first  been 
extracted  by  an  acid  so  as  to  remove  the  lime  salts.  The  wings  or  shells 
are  boiled  with  caustic  alkali  until  they  are  white,  afterward  washed  with 
water,  then  with  dilute  acid  and  water,  and  lastly  extracted  with  alcohol 
and  ether.  If  chitin  so  prepared  is  dissolved  in  cold,  concentrated  sul- 
phuric acid  and  diluted  with  cold  water,  then  pure  chitin  separates  out, 
having  been  set  free  from  the  combination  withthe  other  bodies  (Kraw^kow). 

Hyalin  is  the  chief  organic  constituent  of  the  walls  of  hydatid  cysts.  From  a 
chemical  point  of  view  it  stands  close  to  chitin,  or  between  it  and  protein.  In 
old  and  more  transparent  sacs  it  is  tolerably  free  from  mineral  bodies,  but  in 
younger  sacs  it  contains  a  great  cjuantity  (16  per  cent)  of  lime  salts  (carbonate, 
phosphate,  and  sulphate). 

According  to  Lt'CKE  ^  its  composition  is: 

C        H        N         O 

From  old  cysts 45.3     6.5     5.2     43.0 

From  young  cysts 44.1      6.7     4.5     44.7 


'Araki.  1.  c;  v.  Furth  and  Russo,  Hofmeister;  Beitrage  8. 
*  Virchow's  Arch.,  19. 


688  THE  SKIX   AXD   ITS  SECRETIONS. 

It  differs  from  keratin  on  the  one  hand  and  from  proteids  on  tlie  other  by  the 
absence  of  sulphur,  also  by  its  yielding,  when"  boiled  with  dilute  sulphuric  acid,  a 
variety  of  sugar  in  large  quantities  (50  per  cent),  which  is  reducing,  fermentable, 
and  dextrogyrate.  It  differs  from  chitin  by  tlie  property  of  being  gradually 
dissolved  by' caustic  potash  or  soda,  or  by  dilute  acids;  also  by  its  solubility  on 
heating  with  water  to  150°  C. 

The  coloring  matters  of  the  skin  and  horn-jormations  are  of  different 
kinds,  but  have  not  been  much  studied.  Those  occurring  in  the  stratum 
Malpighii  of  the  skin,  especially  of  the  negro,  and  the  black  or  brown  pig- 
ment occurring  in  the  hair,  belong  to  the  group  of  those  substances  which 
have  received  the  name  melanins. 

Melanins.  This  group  includes  several  different  varieties  of  amorphous 
black  or  brown  pigments  which  are  insoluble  in  water,  alcohol,  ether, 
chloroform,  and  dilute  acids,  and  which  occur  in  the  skin,  hair,  epithelium- 
cells  of  the  retina,  in  sepia,  in  certain  pathological  formations,  and  in  the 
blood  and  urine  in  disease.  Of  these  pigments  there  are  a  few,  such  as  the 
melanin  of  the  eye,  Schmiedeberg's  sarcomelanin,  and  that  from  the 
melanotic  sarcomata  of  horses,  the  hippomelanin  (Nencki,  Sieber,  and 
Berdez),  which  are  soluble  with  difficulty  in  alkalies,  while  others,  such 
as  the  coloring  matter  of  certain  pathological  swellings  in  man,  the 
phynuttorhusin  (Nencki  and  Berdez),  are  readily  soluble  in  alkalies. 
The  humus-like  products,  called  melanoidic  acids  by  Schmiedeberg, 
obtained  on  boiling  proteins  with  mineral  acids,  are  rather  easily  soluble 
in   alkalies. 

Among  the  melanins  there  are  a  few,  for  example  the  choroid  pigment, 
which  are  free  from  sulphur  (Laxdolt  and  others);  others,  on  the  con- 
trary, as  sarcomelanin  and  the  pigment  of  the  hair  and  of  horse-hair,  are 
rather  rich  in  sulphur  (2-4  per  cent),  while  the  phymatorhusin  found  in 
certain  swellings  and  in  the  urine  (Nencki  and  Berdez,  K.  Morner)  is 
very  rich  in  sulphur  (8-10  per  cent).  Whether  any  of  these  pigments, 
especially  the  phymatorhusin,  contains  any  iron  or  not  is  an  important 
though  disputed  point,  for  it  leads  to  the  question  w'hether  these  pigments 
are  formed  from  the  blood-coloring  matters.  According  to  Nencki  and 
Berdez  the  pigment,  phymatorhusin,  isolated  by  them  from  a  melanotic 
sarcoma  did  not  contain  any  iron,  and  according  to  them  is  not  a  deriv- 
ati-^^e  of  haemoglobin.  K.  Morner  and  later  also  Br.^ndl  and  L.  Pfeiffer 
found,  on  the  contrary,  that  this  pigment  did  contain  iron,  and  they 
consider  it  as  a  derivative  of  the  blood-pigments.  The  sarcomelanin 
(from  a  sarcomatous  liver)  analyzed  by  Schmiedeberg  contained  2.7  per 
cent  iron,  which  was  in  organic  combination  in  part  and  could  not  be  com- 
pletely removed  by  dilute  hydrochloric  acid.  The  sarcomelanic  acid  pre- 
pared by  Schmiedeberg  by  the  action  of  alkali  on  this  melanin  contained 
1.07  per  cent  iron.     The  sarcomelanin  investigated  by  Zdarek  and  v. 


MELANINS.  689 

Zeynek  also  contained  0.4  per  cent  iron.  Recently  Wolff'  has  pre- 
pared two  pigments  from  a  melanotic  liver  of  which  one  was  no  doubt 
modified.  The  other,  which  was  soluble  in  a  soda  solution,  contained  2.51 
per  cent  sulphur  and  2.63  per  cent  iron,  which  was  in  great  part  split  off 
by  20  per  cent  hydrochloric  acid.  From  another  liver  he  obtained  on  the 
contrary  a  melanin  free  from  iron  with  1.67  per  cent  sulphur.  From 
this  melanin  he  obtained,  by  treatment  with  bromine,  a  hydro-aromatic 
body  which  was  related  to  xyliton  (a  condensation  product  of  acetone).^ 

The  difficulties  which  attend  the  isolation  and  purification  of  the  mela- 
nins  have  not  been  overcome  in  certain  cases,  while  in  others  it  is  ques- 
tionable whether  the  final  product  obtained  has  not  another  composition 
from  the  original  coloring  matter,  owing  to  the  energetic  chemical  processes 
resorted  to  in  its  purification.  Under  these  circumstances  and  as  no 
doubt  we  have  a  large  number  of  melanins  having  different  composition, 
it  seems  that  a  tabulation  of  the  analyses  of  the  different  melanin  prepa- 
rations can  only  be  of  secondary  importance. 

The  one  or  more  pigments  of  the  human  hair  have  a  low  percentage 
of  nitrogen,  8.5  per  cent  (Sieber),  and  a  variable  but  considerable  amount 
of  sulphur,  2.71-4.10  per  cent.  The  great  quantity  of  iron  oxide  which 
remains  on  incinerating  hair  does  not  seem  to  belong  to  the  pigments- 
The  pigment  of  the  negro's  skin  and  hair  was  found  entirely  free  from  iron 
by  Abel  and  Davis.^  The  pigment  prepared  by  Spiegler  from  the 
hair  of  animals  also  contained  no  iron. 

So  little  is  known  about  the  structural  products  of  the  melanins  or 
melanoids  that  it  is  impossible  to  give  the  origin  of  these  bodies.  As 
undoubtedly  there  are  several  distinct  melanins,  their  origin  must  also  be 
distinct.  The  ferruginous  melanins  should  be  considered  as  originating 
from  the  blood-pigments  until  further  research  proves  otherwise.  Most 
melanins  —  and  this  is  also  true  for  the  melanoids  produced  from  proteins 
on  cleavage  with  acids  (Samuely)  —  yield  indol  or  skatol  and  a  pyrrol 
substance,  and  we  must  therefore  admit  with  Samuely*  that  the  dif- 
ferent chromogen  groups  contained  in  the  protein  molecule,  which  readily 
yield  aromatic  and  specially  heterocyclic  nuclei,  which  condense  with  the 
withdrawal  of  water  and  absorption  of  oxygen,  produce  dark  colored  pro- 
ducts the  mixture  of  which  forms  the  melanoids. 


'  Zdarek  and  v.  Zeynek,  Zeitschr.  f.  physiol.  Chem.,  36;  Wolff,  Hofmeister  Beitrage 
5.  The  literature  on  the  melanins  may  be  found  in  Schmiedeberg,  "Elementarformeln 
einiger  Eiweisskorper,  etc."  Arch.  f.  exp.  Path.  u.  Pliarm.,  39;  also  in  Robert,  Wiener 
Klinik,  27  (1901),  and  Spiegler,  Hofmeister's  Beitrage,  4. 

^  The  summary  of  the  extensive  literature  on  melanotic  pigments  may  be  found  in 
O.  V.  Fiirth,  Centralbl.  f.  allgem.  Path.  u.  Pathol.  Anat.  lo,  1904. 

^  Journ.  of  Expt.  Med.  1,  361. 

*  Hofmeister's  Beitrage,  2. 


690  THE  SKIN  AND  ITS  SECRETIONS. 

It  has  also  been  found  that  by  the  action  of  tyrosinases  upon  tyrosine 
dark  products  similar  to  melanin  are  formed,  and  these,  like  the  animal 
melanins,  yield  substances  smelling  like  skatol  on  fusion  with  alkali. 
Such  a  direct  pigment  formation  caused  by  the  presence  of  tyrosinase  has 
also  been  observed  by  Gessard  in  the  maceration  of  the  skin  of  frogs  and 
toads,  and  certain  investigators,  such  as  Gessard,  v.  Fijrth  and  Schneider,^ 
are  therefore  of  the  opinion  that  tyrosine  is  the  mother-substance  of  the 
melanins. 

In  addition  to  the  coloring  matters  of  the  human  skin  it  is  in  place  here  to 
treat  of  the  pigments  found  in  the  skin  or  epidermal  for  mation  of  animals. 

The  beautiful  color  of  the  feathers  of  many  birds  depends  in  certain  cases  on 
purely  physical  causes  (interference-phenomena),  but  in  other  cases  on  coloring 
matters  of  various  kinds.  Such  a  coloring  matter  is  the  amorphous  reddish-violet 
turacin,  which  contains  7  per  cent  copper  and  whose  spectrum  is  ver}'  similar  to 
that  of  oxyhaemoglobin.  It  must  be  remarked  that  according  to  Laidlaw  ^  turacin 
or  at  least  a  pigment  with  the  same  properties  can  be  obtained  on  boUing  hsemato- 
porphyi-in  in  dilute  ammonia  with  ammoniacal  copper  solution.  KRrKENBERG  * 
found  a  large  number  of  coloring  matters  in  birds'  feathers,  namely,  zooerythrin, 
zoofulvin,  turacoverdin,  zoorubin,  psittacofulvin,  and  others  which  cannot  be 
enumerated  here. 

Tetronerythrin,  so  named  by  Wurm,  is  a  red  amorphous  pigment  which  is 
soluble  in  alcohol  and  ether,  and  which  occurs  in  the  red  warty  spots  over  the  eyes 
of  the  heathcock  and  the  grouse,  and  which  is  verj^  widely  spread  among  the  inver- 
tebrates (Halliburton,  De  Merejkowski,  MacMunn).  Besides  tetronerythrin 
MacMunn  found  in  the  shells  of  crabs  and  lobsters  a  blue  coloring  matter  cyano- 
crystallin,  which  turns  red  with  acids  and  by  boiling  water.  Hmnatoporphyrin, 
according  to  Mac^Iunn,*  also  occurs  in  the  integuments  of  certain  of  the  lower 
animals. 

In  certain  butterflies  (the  pieridinpe)  the  white  pigment  of  the  wings  consists, 
as  shown  by  Hopkins,^  of  uric  acid,  and  the  yellow  pigment  of  a  uric-acid  deriva- 
tive, lepidotic  acid,  which  yields  a  purple  substance,  lepidoporphyrin,  on  warming 
with  dilute  sulphuric  acid.  The  yellow  and  red  pigment  of  the  Vanessa  are, 
according  to  Linden,®  of  an  entirely  different  kind.  In  this  case  we  are  dealing 
with  a  compound  between  proteid  and  a  pigment  which  is  allied  to  bilirubin  or 
urobilin,  i.e.,  a  compound  similar  to  hsemoglobin. 

In  addition  to  the  coloring  matters  thus  far  mentioned  a  few  others  found  in 
certain  animals  (though  not  in  the  skin)  will  be  spoken  of. 

Caxminic  acid,  or  the  red  pigment  of  the  cochineal,  gives  on  oxidation,  accord- 
ing to  LiEBERMANN  and  VoswiNCKEL,'  cochenillic  acid,  CioHgOy,  and  coccinic  acid. 


*  Gessard,  Compt.  rend.  136,  and  Compt.  rend.,  see.  bid.  57;  v.  Furth  and  Schneider, 
Hofmeister's  Beitrage,  1. 

^  Joum.  of  Physiol.  31. 

3  Vergleichende  physiol.  Studien,  Abth.  5,  and  (2.  Reihe)  Abth.  1,  151,  Abth.  2,  1, 
and  Abth.  3,  128. 

*Wunn,  cited  from  Maly's  Jahresber.,  1;  Halliburton,  Joum.  of  Physiol.,  6;  Merej- 
kow.ski,  Compt.  rend.,  93;  MacMunn,  Proc.  Roy.  See,  1883,  and  Joum.  of  Physiol.,  7. 

">  Phil.  Trans.,  186. 

"Pflijger's  Arch.,  98. 

'Ber.  d.  deutsch.  chem.  Gesellsch.,  30. 


SEBUM.  691 

CsHgOfi,  the  first  being  the  tri-carboxyhe  acid,  and  the  other  the  di-carboxylic 
acid  of  m-cresol.  The  beautiful  purple  solution  of  ammonium  carminate  has  two 
absorption-bands  between  D  and  E  which  are  similar  to  those  of  oxyhremoglobin. 
These  bands  lie  nearer  to  E  and  closer  together  and  are  less  sharply  defined.  Pur- 
ple  is  the  evaporated  residue  from  the  purple-violet  secretion,  caused  by  the  action 
of  the  sunlight,  from  the  so-called  "purple  gland"  of  the  mantle  of  certain  species 
of  murex  and  purpura.     Its  chemical  nature  has  not  been  investigated. 

Among  the  remaining  coloring  matters  found  in  invertebrates  may  be  men- 
tioned him  stentorin,  actiniochrom,  honellin,  poly  per  ythri?i,  pentacrinin,  antedonin, 
crustaceorubin,  janthinin,  and  chlorophyll. 

Sebum  when  freshly  secreted  is  an  oily  semi-fluid  mass  which  solidifies 
on  the  upper  surface  of  the  skin,  forming  a  greasy  coating.  Sebum  is 
according  to  Rohmann  and  Linser  a  mixture  of  the  secretion  of  the 
sebaceous  glands  and  of  the  constituents  of  the  epidermis.  Hoppe- 
Seyler  has  found  in  the  sebum  a  body  similar  to  casein  besides  albumin 
and  fat.  According  to  Rohmann  and  Linser  true  fat  occurs  only  to  a 
very  slight  extent.  On  saponification  the  sebum  gives  an  oil,  dcrmolein, 
which  combines  readily  with  iodine,  and  another  body,  dermocerin,  which 
melts  at  64-65°  and  which  occurs  to  a  considerable  extent  in  dermoid 
cysts  and  which  is  perhaps  identical  with  the  constituent  of  cysts  called 
cetylalcohol  by  v.  Zeynek.  The  amount  of  cholesterin  in  this  secretion 
is  small  and  originates  essentially  from  the  epidermoidal  formation. 
Cholesterin  is  found  in  especially  large  quantities  in  the  vernix  caseosa. 
The  solids  of  the  sebum  consist  chiefly  of  fat,  epithelium-cells,  and  protein 
bodies;  the  vernix  caseosa  is  made  up  chiefly  of  fat.  Rltppel  ^  found  on 
an  average  in  the  vernix  caseosa  348.52  p.  m.  water  and  138.72  p.  m. 
ether  extractives.     Besides  cholesterin  he  found  also  isocholesterin. 

On  account  of  the  generally  diffused  view  that  the  wax  of  the  plant 
epidermis  serves  as  protection  for  the  inner  parts  of  the  fruit  and  plant, 
Liebreich  ^  has  suggested  that  these  combinations  of  fatty  acids  with 
monatomic  alcohols  are  the  cause  of  the  waxes  having  a  greater  resistance 
as  compared  with  the  glycerine  fats.  He  also  considers  that  the  choles- 
terin fats  play  the  role  of  a  protective  fat  in  the  animal  kingdom,  and  he 
has  been  able  to  detect  cholesterin  fat  in  human  skin  and  hair,  in  vernix 
caseosa,  whalebone,  tortoise-shell,  cow's  horn,  the  feathers  and  beaks  of 
several  birds,  the  spines  of  the  hedgehog  and  porcupine,  the  hoofs  of 
horses,  etc.  He  draws  the  following  conclusion  from  this,  namely,  that 
the  cholesterin  fats  always  appear  in  combination  with  the  keratinous 
substance,  and  that  the  cholesterin  fat,  like  the  wax  of  plants,  serves  as 
protection  for  the  skin-surface  of  animals. 

'  Hoppe-Seyler,  Physiol.  Chem.  760;  Linser  with  Rohmann,  Centralbl.  f.  Physiol. 
19,  317;  see  also  reference  in  ibid.  18  from  Deutsch.  Arch.  f.  klin.  Med.,  1904;  Riippel 
Zeitschr.  f.  physiol.  Chem.,  21. 

'  Virchow's  Arch.,  121. 


692  THE   SKIX  AXD   ITS   SECRETIONS. 

In  the  fatt}''  protective  substance  secreted  by  the  Psylla  alni  Sundvik  '  has 
found  psylla-alcohol,  CajHggO,  which  exists  there  as  an  ester  in  combination  with 
psyllic  acid,  CgaHesCOOH. 

Cenunen  is  a  mixture  of  the  secretion  of  the  sebaceous  and  sweat  glands 
of  the  cartilaginous  part  of  the  outer  passages  of  the  ear.  It  contains 
chiefly  soaps  and  fat,  fatt}^  acids,  cholesterin  and  proteid,  and  besides 
these  a  red  substance  easily  soluble  in  alcohol  and  ^yith  a  bitter-sweet 
taste. - 

The  preputial  secretion,  smegma  pneputii,  contains  chiefly  fat,  also 
cholesterin  and  ammonium  soaps,  which  probably  are  produced  from 
decomposed  urine.  The  hippuric  acid,  benzoic  acid,  and  calcium  oxalate 
found  in  the  smegma  of  the  horse  have  probably  the  same  origin. 

We  may  also  consider  as  a  preputial  secretion  the  castoreum,  which  is  secreted 
by  two  peculiar  glandular  sacs  in  the  prepuce  of  the  beaver.  The  castoreum  is  a 
mixture  of  proteins,  fat,  resins,  traces  of  phenol  (volatile  oil),  and  a  non-nitrog- 
enous body,  castorin,  crystallizing  in  four-sided  needles  from  alcohol,  insoluble 
in  cold  water,  but  somewhat  soluble  in  boiling  water,  and  whose  comj^osition  is 
little  known. 

In  the  secretion  from  the  anal  glands  of  the  skunk  butyl  mercaptan  and  alkyl 
sulphide  have  been  found  (Aldrich,  E.  Beckmaxn  ^). 

Wool-fat,  or  the  so-called  fat-sweat  of  sheep,  is  a  mixture  of  the  secretion  of 
the  sudoriparous  and  sebaceous  glands.  There  is  found  in  the  watery  extract  a 
large  quantity  of  potassium  which  is  combined  with  organic  acid,  volatile  and  non- 
volatile fatty  acids,  benzoic  acid,  phenol-sulphuric  acid,  lactic  acid,  malic  acid, 
succinic  acid,  and  others.  The  fat  contains,  among  other  bodies,  abundant  quan- 
tities of  ethers  of  fatty  acids  with  cholesterin  and  isocholesterin.  Darmstadter 
and  LiFSCHxJTZ  have  found  other  alcohols  in  wool-fat  besides  mjTistic  acid,  also 
two  oxyfatty  acids,  lanoceric  acid,  C3oH6g04,  and  lanopalmitic  acid,  C.eHgjOa. 
According  to  Rohmaxn  *  wool-fat  contains  a  body  lanocerin,  which  is  the  internal 
anhydride  of  the  above-mentioned  lanoceric  acid.  Lanocerin  is  obtained  without 
saponification  by  repeatedly  boiling  lanolin  with  method  alcohol,  dissolving  the 
insoluble  residue  in  ether  and  precipitating  with  alcohol. 

The  secretion  of  the  coccygeal  glands  of  ducks  and  geese  contains  a  body  similar 
to  casein,  besides  albumin,  nuclein,  lecithin,  and  fat,  but  no  sugar  (De  Jonge). 
The  chief  constituent  is  octadecyl  alcohol,  Ci,H3,0,  which  represents  40-45  per  cent 
of  the  ethereal  extract  (Rohmaxx).  The  fatty  acids  are  oleic  acid,  small  amounts 
of  caprylic  acid,  palmitic  acid,  and  stearic  acid,  and  optical  i-somers  of  lauric  and 
myristic  acid.  The  fatty  acids  are  in  great  part  combined  with  the  octadecylic 
acid,  and  this  is  probably  formed  by  the  reduction  of  stearic  acid  or  oleic  acid. 
The  secretion  also  contains  a  substance  related  to  lanocerin  which  Rohmann  calls 
pennacerin.  Poisonous  bodies  have  been  found  in  the  secretion  of  the  skin  of  the 
salamander  and  the  toad,  namely,  samandarin  (Zaleski,  Faust)  and  bufidia 
(Jorxara  and  Casali),  hufotalin  and  the  disputed  bodies  hujonin  and  Imjotenin 


*  Zeitschr.  f.  physiol.  Chem.,  17,  25,  and  32. 

^  See  Lamois  and  Martz,  Maly's  .Jahresber.,  27,  40. 

'Aldrich,  Joum.  of  Expt.  Med.,  1;  Beckmann,  Maly's  Jahresber.,  26,  566. 

*  Darmstadter  and  Lifschtitz,  Ber.  d.  d.  Chem.,  Gesellsch,  29  and  31;  Rohmann, 
Hofmeisters  Beitrage  5  and  Centrabll.  f.  Physiol.  19,  317. 


PERSPIRATION.  693 

(Faust,  Bertrand  and  Phisalix  ').     Thalassin  is  the  crystalline  body  discovered 
by  RiCHET  '  which  is  the  poisonous  constituent  of  the  feelers  of  the  sea  nettle. 

The  Perspiration.  Of  the  secretions  of  the  skin,  whose  quantity 
amounts  to  about  g^j  of  the  weight  of  the  body,  a  disproportionally  large 
part  consists  of  water.  Next  to  the  kidneys,  the  skin  in  man  is  the  most 
important  means  for  the  elimination  of  water.  As  the  glands  of  the  skin 
and  the  kidneys  stand  near  to  each  other  in  regard  to  their  functions, 
they  may  to  a  certain  extent  act  vicariously. 

The  circumstances  which  influence  the  secretion  of  perspiration  are 
very  numerous,  and  the  quantity  of  sweat  secreted  must  consequently 
vary  considerably.  The  secretion  differs  for  different  parts  of  the  skin, 
and  it  has  been  stated  that  the  perspiration  of  the  cheek,  that  of  the  palm 
of  the  hand,  and  that  under  the  arm  stand  to  each  other  as  100:90:45- 
From  the  unequal  secretion  on  different  parts  of  the  body  it  follows  that 
no  results  as  to  the  quantity  of  secretion  for  the  entire  surface  of  the  body 
can  be  calculated  from  the  quantity  secreted  by  a  small  part  of  the  skin  in 
a  given  time.  In  determining  the  total  quantity  a  stronger  secretion  is 
as  a  rule  produced,  and  as  the  glands  can  with  difficulty  work  for  a  long 
time  with  the  same  energy,  it  is  hardly  correct  to  estimate  the  quantity 
of  secretion  per  day  from  a  strong  secretion  during  only  a  short  time. 

The  perspiration  obtained  for  investigation  is  never  quite  pure,  but 
contains  cast-off  epidermis-cells,  also  cells  and  fat-globules  from  the  seba- 
ceous glands.  Filtered  perspiration  is  a  clear,  colorless  fluid  with  a  salty 
taste  and  of  different  odors  from  different  parts  of  the  body.  The  physio- 
logical reaction  is  acid,  according  to  most  statements.  Under  certain 
conditions  also  an  alkaline  sweat  may  be  secreted  (Trumpy  and  Luch- 
sixGER,  Heuss).  An  alkaline  reaction  may  also  depend  on  a  decompo- 
sition with  the  formation  of  ammonia.  According  to  a  few  investigators 
the  physiological  reaction  is  alkaline,  and  an  acid  reaction  depends, 
according  to  them,  upon  an  admixture  of  fatt}'  acids  from  the  sebum. 
Camerer  found  that  the  reaction  of  human  perspiration  in  certain  cases 
was  acid  and  in  others  alkaline.  ^^Ioriggia  found  that  the  sweat  from 
herbivora  was  ordinarily  alkaline,  while  that  from  carnivora  was  gener- 
ally acid.     According  to  Smith ^   horse's  sweat  is  strongly  alkaline. 

'  De  Jonge,  Zeitschr.  f.  physiol.  Chem.,  3;  Roliniann  1.  c;  Zaleski,  Hoppe-Seyler's 
Med.-chem.  Untersuch.,  85;  Faust,  Arch.  f.  exp.  Path.  u.  Phami.,  -tl;  Jomara  and  Casali, 
Maly's  Jahresber.,  3;  Faust,  Arch.  f.  exp.  Path.  u.  Pharm.,  47  and  49;  Bertrand,  Compt. 
rend.,  135;  Bertrand  and  Phisalix,  ibid. 

2  Pfliiger's  Arch.,  108. 

^  Trumpy  and  Luchsinger,  Pfliiger's  Arch.,  18;  Ileuss,  Maly's  Jahresber.,  22;  Camerer, 
Zeitschr.  f.  Biologic,  41;  Moriggia,  Moleschott's  Untersuch.  zur  Xaturlehre,  11;  Smith, 
Journ.  of  Physiol.,  11.  In  regard  to  the  older  literature  on  perspiration,  see  Hermann's 
Handbuch,  5,  Thl.  1,  421  and  543. 


694  THE  SKIN  AND  ITS  SECRETIONS. 

The  specific  gravity  of  human  perspiration  varies  between  1.001  and 
1.010.  It  contains  977.4r-995.6  p.  m.,  average  about  982  p.  m.  water. 
The  soUds  are  4.4-22.6  p.  m.  The  molecular  concentration  is  also  very 
variable  and  the  freezing-point  depression  depends  essentially  upon  the 
content  of  NaCl.  ARDix-DELTEiLfound  A=  -0.08-0.46°,  average -0.237°. 
Brieger  and  Disselhorst  ^  found  with  perspiration  containing  2.9,  7.07 
and  13.5  p.  m.  NaCl,  that  the  A  was  equal  to  -  0.322°,  -  0.608°  and  -  1.002°, 
respectively.  The  organic  bodies  are  neutral  fats,  cholesterin,  volatile  fatty 
acids,  traces  of  jprotein  (according  to  Leclerc  and  Smith  always  in  horses, 
and  according  to  Gaube  regularly  in  man,  while  Leube  ^  claims  only 
sometimes  after  hot  baths,  in  Bright's  disease,  and  after  the  use  of  pilo- 
carpin),  also  creatinine  (Capraxica),  aromatic  oxyacids,  ethereal- sulphuric 
acids  of  -phenol  and  skatoxyl  (Kast^),  sometimes  also  of  indoxyl,  and 
lastly  urea.  The  quantity  of  urea  has  been  determined  by  Argutinsky. 
In  two  steam-bath  experiments,  in  which  in  the  course  of  h  and  |  hour 
respectively  he  obtained  225  and  330  c.  c.  of  perspiration,  he  found  1.61 
and  1.24  p.  m.  urea.  Of  the  total  nitrogen  of  the  perspiration  in  these 
two  experiments  68.5  per  cent  and  74.9  per  cent  respectively  belong  to 
the  urea.  From  Argutinsky's  experiments,  and  also  from  those  of 
Cramer,^  it  follows  that  of  the  total  nitrogen  a  portion  not  to  be  disre- 
garded is  eliminated  by  the  perspiration.  This  portion  was  indeed  12 
per  cent  in  an  experiment  of  Cramer  at  high  temperature  and  powerful 
muscular  activity.  Cramer  has  also  found  ammonia  in  the  perspiration. 
In  uraemia,  and  in  anuria  in  cholera,  urea  may  be  secreted  in  such  quan- 
tities by  the  sweat-glands  that  crystals  deposit  upon  the  skin.  The  mineral 
bodies  consist  chiefiy  of  sodium  chloride  w^ith  some  potassium  chloride, 
alkali  sulphate,  and  phosphate.  The  relative  quantities  of  these  in  per- 
spiration differ  materially  from  the  quantities  in  the  urine  (Favre,^  Kast). 
The  relationship,  according  to  Kast,  is  as  follows: 


Chlorine 

In  perspiration 1 

In  urine 1 


Phosphate    :   Sulphate 
0.0015     :     0.009 
0.1320    :     0.397 


Kast  found  that  the  proportion  of  ethereal-sulphuric  acid  to  the  sul- 
phate-sulphuric acid  in  perspiration  was  1:12.     After  the  administration 


^  Ardin-Delteil,  Maly's  Jahresber.,  30;  Brieger  and  Disselhorst,  Deutsch.  med.  Woch- 
encshr.,  29. 

^Leclerc,  Compt.  rend.,  107;  Gaube,  Maly's  Jahresber.,  22;  Leube,  Virchow's  Arch., 
48  and  50,  and  Arch.  f.  klin.  Med.,  7. 

^  Capranica,  Maly's  Jahresber.,  12;  Kast,  Zeitschr.  f.  physiol.  Chem.,  11. 

^Argutinsky,  Pfliiger's  Arch.,  46;  Cramer,  Arch.  f.  Hygiene,  10. 

*  Compt.  rend.,  35.  and  Arch,  g^ner.  de  Meu.  (5),  2. 


EXCHANGE  OF  GAS  THROUGH  THE  SKIN.  695 

of  aromatic  substances  the  ethereal-sulphuric  acid  does  not  increase  to 
the  same  extent  in  the  perspiration  as  in  the  urine  (see  Chapter  XV). 

Sugar  may  pass  into  the  perspiration  in  diabetes,  but  the  passage  of  the  bile- 
coloring  matters  has  not  been  positively  shown  in  this  secretion.  Benzoic  acid, 
succinic  acid,  tartaric  acid,  iodine,  arsenic,  mercuric  chloride,  and  quinine  pass 
into  the  perspiration.  Uric  acid  has  also  been  found  in  the  perspiration  in  gout 
and  cystine  in  cystinuria. 

Chromhidrosis  is  the  name  given  to  the  secretion  of  colored  perspiration. 
Sometimes  perspiration  has  been  observed  to  be  colored  blue  by  indigo  (Bizio),  by 
pyocyanin,  or  by  ferro-pho.sphate  (Kollmann').  True  blood-sweat,  in  which 
blood-corpuscles  exude  from  the  opening  of  the  glands,  has  also  been  observed. 

The  exchange  of  gas  through  the  skin  in  man  is  of  very  little  importance 
compared  with  the  exchange  of  gas  by  the  lungs.  The  absorption  of 
oxygen  by  the  skin,  which  was  first  shown  by  Regxault  and  Reiset 
is  very  small,  and  according  to  Zuelzer  amounts  under  the  most  favor- 
able circumstances  to  j-Jo  of  the  oxygen  absorbed  by  the  lungs.  The 
quantity  of  carbon  dioxide  eliminated  by  the  skin  increases  with  the  rise 
of  temperature  (Aubert,  Rohrig,  Fubixi  and  Ronchi,  Barratt^),  It 
is  also  greater  in  light  than  in  darkness.  It  is  greater  during  digestion 
than  when  fasting,  and  greater  after  a  vegetable  than  after  an  animal 
diet  (FuBiNi  and  Ronchi).  The  quantity  calculated  by  various  inves- 
tigators for  the  entire  skin  surface  in  twenty-four  hours  varies  between 
2.23  and  32.8  grams.^  In  a  horse,  Zuntz  with  Lehmanx  and  Hagemann,* 
found  for  twenty-four  hours  an  elimination  of  carbon  dioxide  by  the  skin 
and  intestine  which  amounted  to  nearly  3  per  cent  of  the  total  respiration. 
Less  than  four-fifths  of  this  carbon  dioxide  came  from  the  skin  respira- 
tion. According  to  the  same  investigators  the  skin  respiration  equals 
2^  per  cent  of  the  simultaneous  lung  respiration. 


'  Bizio,  Wien.  Sitzungsber.,  39;  KoUmann,  cited  from  v.  Gorup-Besanez's  Lehrbuch, 
4.  Aufl.,  5.55. 

^Zuelzer,  Zeitschr.  f.  klin.,  Med.,  53;  Aubert,  Pfliiger's  Arch.,  6;  Rohrig,  Deutsch. 
Klin.,  1872,  209;  Fubini  and  Ronchi,  Moleschott's  Untersuch.  z.  Naturlehre,  12;  Barratt, 
Joum.  of  Physiol.,  21. 

^See  Hoppe-Seyler,  Physiol.  Chem.,  580. 

*  Arch.  f.  (Anat.,  u.).  Physiol.,  1894,  and  Maly's  Jahresber.,  24. 


CHAPTER  XVII. 

CHEMISTRY  OF  RESPIRATION. 

During  life  a  constant  exchange  of  gases  takes  place  between  the 
animal  body  and  the  surrounding  medium.  Oxygen  is  inspired  and 
carbon  dioxide  expired.  This  exchange  of  gases,  which  is  called  respira- 
tion, is  brought  about  in  man  and  vertebrates  by  the  nutritive  fluids, 
blood  and  lymph,  which  circulate  in  the  body  and  which  are  in  constant 
comm.unication  with  the  outer  medium  on  one  side  and  the  tissue-elements 
on  the  other.  Such  an  exchange  of  gaseous  constituents  may  take  place 
wherever  the  anatomical  conditions  offer  no  obstacle,  and  in  man  it  may 
go  on  in  the  intestinal  tract,  through  the  skin,  and  in  the  lungs.  As 
compared  with  the  exchange  of  gas  in  the  lungs,  the  exchange  already 
mentioned,  which  occurs  in  the  intestine  and  through  the  skin,  is  very 
insignificant.  For  this  reason  we  will  discuss  in  this  chapter  only  the 
exchange  of  gas  between  the  blood  and  the  air  of  the  lungs  on  one  side 
and  the  blood  and  lymph  and  the  tissues  on  the  other.  The  first  is  often 
designated  as  external  respiration,  and  the  other,  internal  respiration. 

I.    The  Gases  of  the  Blood. 

Since  the  pioneer  investigations  of  Magnus  and  Lothar  Meyer  the 
gases  of  the  blood  have  formed  the  subject  of  repeated  careful  investiga- 
tions by  prominent  experimenters,  among  whom  must  be  mentioned  first 
C.  LuDwiG  and  his  pupils  and  E.  Pfluger  and  his  school.  By  these 
investigations  not  only  has  science  been  enriched  by  a  mass  of  facts,  but 
also  the  methods  themselves  have  been  made  more  perfect  and  accurate. 
In  regard  to  these  methods,  as  also  in  regard  to  the  laws  of  the  absorption 
of  gases  by  liquids,  dissociation,  and  related  questions,  the  reader  is  referred 
to  text-books  on  physiology,  on  physics,  and  on  gasometric  analysis. 

The  gases  occurring  in  blood  under  physiological  conditions  are  oxygen, 
carbon  dioxide  and  nitrogen,  and  traces  of  argon,  hydrogen,  hydrocarbons 
and  carbon  monoxide.  The  nitrogen  is  found  only  in  very  small  quan- 
tities, on  an  average  1.2  vols,  per  cent.  The  quantity  is  here,  as  in  all 
following  experiments,  calculated  for  0°  C.  and  760  mm.  pressure.  The 
nitrogen  seems  to  be  simply  absorbed  by  the  blood,  at  least  in  great  part. 

696 


GASES  OF  THE   BLOOD.  697 

It  appears,  like  argon,  to  play  no  direct  part  in  the  processes  of  life,  and 
its  quantity  varies  but  slightly  in  the  blood  of  different  blood-vessels. 

The  oxygen  and  carbon  dioxide  behave  otherwise,  as  their  quantities 
have  significant  variations,  not  only  in  the  blood  from  different  blood- 
vessels, but  also  because  many  conditions,  such  as  a  difference  in  the 
rapidity  of  circulation,  a  different  temperature,  rest  and  activity,  cause  a 
change.  In  regard  to  the  gases  they  contain  the  greatest  difference  is 
observable  between  the  blood  of  the  arteries  and  that  of  the  veins. 

The  quantity  of  oxygen  in  the  arterial  blood  of  dogs  is  on  an  average 
22  vols,  per  cent  (Pfluger).  In  human  blood  Setschenow  found  about 
the  same  quantity,  nameh%  21.6  vols,  per  cent.  Lower  figures  have  been 
found  for  rabbit's  and  bird's  blood,  respectively  13.2  per  cent  and  10-15 
per  cent  (Walter  Jolyet).  Venous  blood  in  different  vascular  regions 
has  very  variable  quantities  of  oxygen.  By  summarizing  a  great  num- 
ber of  analyses  by  different  experimenters  Zuntz  has  calculated  that  the 
venous  blood  of  the  right  side  of  the  heart  contains  on  an  average  7.15 
per  cent  less  oxygen  than  the  arterial  blood. 

The  quantity  of  carbon  dioxide  in  the  arterial  blood  (of  dogs)  is  about 
40  vols,  per  cent  (Ludwig,  Setschenow,  PflIjger,  P.  Bert,  Bohr  and 
Henriques  and  others),  or  a  little  above.  Setschenow  found  40.3  vols, 
per  cent  in  human  arterial  blood.  The  quantity  of  carbon  dioxide  in 
venous  blood  varies  still  more  (Ludwig,  PFLiJGER,  and  their  pupils, 
P.  Bert,  Mathieu  and  Urbain,  and  others).  According  to  the 
calculations  of  Zuntz,  the  venous  blood  of  the  right  side  of  the 
heart  contains  about  8.2  per  cent  more  carbon  dioxide  than  the 
arterial.  The  average  amount  may  be  put  down  as  50  vols,  per  cent. 
Holmgren  found  in  blood  after  asphyxiation  even  69.21  vols,  per  cent 
carbon  dioxide.^ 

Oxygen  is  absorbed  only  to  a  small  extent  by  the  plasma,  which  only 
absorbs  0.65  per  cent  oxygen.  The  greater  part  or  nearly  all  of  the 
oxygen  is  loosely  combined  with  the  haemoglobin.  The  quantity  of 
oxygen  which  is  contained  in  the  blood  of  the  dog  corresponds  closely  to 
the  quantity  which  from  the  activity  of  the  haemoglobin  we  should  expect 
to  combine  with  oxygen,  and  from  the  quantity  of  haemoglobin  contained 
therein.  It  is  difficult  to  ascertain  how  far  the  circulating  arterial  blood 
is  saturated  with  oxygen,  as  immediately  after  bleeding  a  loss  of  oxygen 
always  takes  place.  Still  it  seems  to  be  unquestionable  that  it  is  not 
quite  completely  saturated  with  oxygen  in  life. 

'  Afl  the  figures  given  above  may  be  found  in  Zuntz's  "Die  Gase  des  Blutes"  in 
Hermann's  Handbuch  d.  Pfiysiol.,  4,  Thl.  2,  33-43,  which  also  contains  detailed  state- 
ments and  the  pertinent  literature,  and  Bohr  in  Nagel's  Handbuch  der  Physiologie  des 
Menschen,  Bd.  1.  Hefte  1,  1905. 


698  CHEMISTRY   OF  RESPIRATION. 

The  carbon  dioxide  of  the  blood  occurs  in  part,  and  indeed,  according 
to  the  investigations  of  Alex.  Schmidt,^  Zuntz,^  and  L.  Fredericq,^  to 
the  extent  of  at  least  one-third  in  the  blood-corpuscles,  also  in  part,  and 
in  fact  the  greatest  part,  in  the  plasma  or  serum.  According  to  Bohr  *  a 
pressure  of  about  30  mm.  may  be  considered  as  the  average  pressure  of 
the  carbon  dioxide  in  the  organism,  and  with  such  a  pressure  the  quantity 
of  physically  dissolved  CO2  in  100  c.c.  of  the  blood  amounts  to  2.01  c.c. 
As  the  blood  with  this  tension  takes  up  about  40  vols,  per  cent  CO,,  hence 
about  5  per  cent  of  the  total  carbon  dioxide  is  simply  dissolved.  Under 
the  assumption  that  the  blood  corpuscles  make  up  about  J  of  the  volume 
of  the  blood,  of  the  physically  dissolved  CO2,  0.59  c.c.  exists  with  the 
corpuscles  and  1.42  c.c.  with  the  plasma. 

As  the  blood  corpuscles  in  100  c.c.  blood  as  above  stated  take  up  at 
the  above  pressure  about  14  c.c.  COj  only  a  small  part  of  its  COj  is  physi- 
cally dissolved.  The  chief  mass  of  the  COj  is  loosely  combined  and  the 
constituent  of  these  cells  which  unites  with  the  CO,  seems  to  be  the  alkali 
combined  with  phosphoric  acid,  oxyhsemoglobin  or  haemoglobin,  and 
globulin  on  one  side  and  the  haemoglobin  itself  on  the  other.  That  in  the 
red  blood-corpuscles  alkali  phosphate  occurs  in  such  quantities  that  it 
may  be  of  importance  in  the  combination  with  carbon  dioxide  is  not  to 
be  doubted;  and  it  must  be  allowed  that  from  the  diphosphate,  by  a 
greater  partial  pressure  of  the  carbon  dioxide,  monophosphate  and  alkali 
carbonate  are  formed,  while  by  a  lower  partial  pressure  of  the  carbon 
dioxide  the  mass  action  of  the  phosphoric  acid  comes  again  into  play,  so 
that,  with  the  carbon  dioxide  becoming  free,  a  re-formation  of  alkali 
diphosphate  takes  place.  It  is  generally  admitted  that  the  blood-coloring 
matters,  especially  the  oxyhaemoglobin  which  can  expel  carbon  dioxide 
from  sodium  carbonate  in  vacuo,  acts  like  acids;  and  as  the  globulins  also 
act  similarly  (see  below),  these  bodies  may  also  occur  in  the  blood- 
corpuscles  as  an  alkali  combination.  The  alkali  of  the  blood-corpuscles 
must  therefore,  according  to  the  law  of  mass  action,  be  divided  between 
the  carbon  dioxide,  phosphoric  acid,  and  the  other  constituents  of  the 
blood-corpuscles  which  possess  acidic  properties,  and  among  these  espe- 
cially the  blood  pigments,  because  the  globulin  can  hardly  be  of  importance 
on  account  of  its  small  quantity.  By  greater  mass  action  or  greater 
partial  pressure  of  the  carbon  dioxide,  bicarbonate  must  be  formed  at 
the  expense  of  the  diphosphates  and  the  other  alkali  combinations,  while 
at  a  diminished  partial  pressure  of  the  same  gas,  with  the  escape  of  carbon 

*  Ber.  d.  k.  siichs.  Gesellsch.  d.  Wissensch.,  math.-phys.  Klasse,  1867. 
'Centralbl.  f.  d.  med.  Wissensch.,  1867,  529. 

'  Recherches  sur  la  constitution  du  Plasma  sanguin,  1878,  50,  51. 

*  In  regard  to  the  work  of  Bohr  we  will  refer  here  and  in  future  to  Nagel's  Handbuch 
der  Physiologie  des  Menschen,  Bd.  1,  Hefte  1. 


THE   CARBON   DIOXIDE  OF  THE   BLOOD.  699 

•dioxide,  the  alkali  diphosphate  and  the  other  alkali  combinations  must 
be  re-formed  at  the  cost  of  the  bicarbonate. 

Haemoglobin  must  nevertheless,  as  the  investigations  of  Setschenow  * 
and  ZuNTz,  and  especially  those  of  Bohr  and  Torup,^  have  shown,  be 
able  to  hold  the  carbon  dioxide  loosely  combined  even  in  the  absence  of 
alkali.  Bohr  has  also  found  that  the  dissociation  curve  of  the  carbon- 
dioxide  haemoglobin  corresponds  essentially  to  the  curve  of  the  absorp- 
tion of  carbon  dioxide,  on  which  ground  he  and  Torup  consider  the  haemo- 
globin itself  as  of  importance  in  the  binding  of  the  carbon  dioxide  of  the 
blood,  and  not  its  alkali  combinations.  According  to  Bohr  the  haemo- 
globin takes  up  the  two  gases,  oxygen  and  carbon  dioxide,  simultane- 
ously by  the  oxygen  uniting  with  the  pigment  nucleus  and  the  carbon 
dioxide  with  the  protein  component.  But  as  according  to  the  researches 
of  ZuNTz  ^  the  combination  of  haemoglobin  with  the  alkali  is  first  split 
to  any  great  extent  with  a  carbon  dioxide  tension  of  more  than  70  mm.,  it 
must  be  admitted  that  with  the  ordinary  CO2  pressure  in  the  organism,  the 
combination  of  the  carbon  dioxide  in  the  blood  corpuscles  does  not  essen- 
tially take  place  through  the  agency  of  the  alkali  but  chiefly  by  means 
of  the  haemoglobin. 

The  chief  part  of  the  carbon  dioxide  of  the  blood  is  found  in  the 
blood-plasma  or  the  blood-serum,  which  follows  from  the  fact  that  the 
serum  is  richer  in  carbon  dioxide  than  the  corresponding  blood  itself. 
By  experiments  with  the  air-pump  on  blood-serum  it  has  been  found 
that  the  chief  part  of  the  carbon  dioxide  contained  in  the  serum  is  given 
off  in  a  vacuum,  while  a  smaller  part  can  be  removed  only  after  the 
addition  of  an  acid.  The  red  blood-corpuscles  also  act  as  an  acid, 
and  therefore  in  blood  all  the  carbon  dioxide  is  expelled  in  vacuo. 
Hence  a  part  of  the  carbon  dioxide  is  in  firm  chemical  combination 
in  the  serum. 

Absorption  experiments  with  blood-serum  have  shown  us  further  that 
the  carbon  dioxide  which  can  be  pumped  out  is  in  great  part  looselv  chem- 
ically combined,  and  from  this  loose  combination  of  the  carbon  ^dioxide 
it  necessarily  follows  that  the  serum  must  also  contain  simplv  absorbed 
carbon  dioxide.  For  the  form  of  binding  of  the  carbon  dioxide  contained 
m  the  serum  or.  the  plasma  there  are  the  three  following  possibilities: 
1.  A  part  of  the  carbon  dioxide  is  simply  absorbed;  2.  Another  part  is 
in  loose  chemical  combination;  3.  A  third  part  is  in  firm  chemical  com- 
bination. 


>  Centralbl.  f.  d.  med.  Wissensch.,  1877.     See  also  Zuntz  in  Hermann's  Handbuch 
76.  ' 

2  Zuntz,  1.  c,  76;  Bohr,  Maly's  Jahresber.,  17;  Torup,  ibid. 
'Centralbl.  f.  d.  med.  Wissensch.,  1867. 


700  CHEMISTRY  OF  RESPIRATION. 

The  quantity  of  physically  dissolved  carbon  dioxide  in  the  serum  cannot 
be  higher  than  about  2  vols,  per  cent,  as  the  quantity  of  carbon  dioxide  in 
the  plasma  corresponding  to  100  c.c.  of  blood  is  given  above  as  1.42  c.c. 
The  quantity  of  carbon  dioxide  in  the  blood-serum  which  is  combined 
as  a  firm  chemical  union  depends  upon  the  quantity  of  simple  alkali 
carbonate  in  the  serum.  This  amount  is  not  known,  and  it  cannot  be 
determined  either  by  the  alkalinity  found  by  titration,  nor  can  it  be  calcu- 
lated from  the  excess  of  alkali  found  in  the  ash,  because  the  alkali  is  not 
only  combined  with  carbon  dioxide,  but  also  with  other  bodies,  especially 
with  protein.  The  quantity  of  carbon  dioxide  in  firm  chemical  combi- 
nation cannot  be  ascertained  after  pumping  out  in  vacuo  without  the 
addition  of  acid,  because  to  all  appearances  certain  active  constituents 
of  the  serum,  acting  like  acids,  expel  carbon  dioxide  from  the  simple 
carbonate.  The  quantity  of  carbon  dioxide  not  expelled  from  dog- 
serum  by  vacuum  alone  without  the  addition  of  acid  amounts  to  4.9  to 
9.3  vols,  per  cent,  according  to  the  determinations  of  Pfluger.^ 

From  the  occurrence  of  simple  alkali  carbonates  in  the  blood-serum  it 
naturally  follows  that  a  part  of  the  loosely  combined  carbon  dioxide  of 
the  serum  which  can  be  pumped  out  must  exist  as  bicarbonate.  The 
occurrence  of  this  combination  in  the  blood-serum  has  also  been  directly 
shown.  In  experiments  with  the  pump,  as  well  as  in  absorption  experi- 
ments, the  serum  behaves  in  other  ways  different  from  a  solution  of  bicar- 
bonate, or  carbonate  of  a  corresponding  concentration;  and  the  behavior 
of  the  loosely  combined  carbon  dioxide  in  the  serum  can  be  explained 
only  by  the  occurrence  of  bicarbonate  in  the  serum.  By  means  of 
vacuum  the  serum  always  allows  much  more  than  one  half  of  the  carbon 
dioxide  to  be  expelled,  and  it  follows  from  this  that  in  the  pumping  out 
not  only  may  a  dissociation  of  the  bicarbonate  take  place,  but  also  a 
conversion  of  the  double  sodium  carbonate  into  a  simple  salt.  As  we 
know  of  no  other  carbon-dioxide  combination  besides  the  bicarbonate 
in  the  serum  from  which  the  carbon  dioxide  can  be  set  free  by  simple 
dissociation  in  vacuo,  it  must  be  assumed  that  the  serum  contains  other 
weak  acids,  in  addition  to  the  carbon  dioxide,  which  contend  with  it  for 
the  alkalies,  and  which  expel  the  carbon  dioxide  from  simple  carbonates 
in  vacuo.  The  carbon  dioxide  which  is  expelled  by  means  of  the  pump, 
and  which,  without  regard  to  the  quantity  merely  absorbed,  is  generally 
designated  as  "  carbon  dioxide  in  loose  chemical  combination,"  is  thus 
only  obtained  in  part  in  dissociable  loose  combinations;  in  part  it  origi- 
nates from  the  simple  carbonates,  from  which  it  is  expelled  in  vacuo  by 
other  weak  acids. 

» E.  Pfluger,  Ueber  die  Kohlensaure  des  Blutes,  Bonn,  1864,  11.     Cited  from  Zuntz 
in  Hermann's  Handbuch,  65. 


COMBINATIONS   OF   THE   CARBON   DIOXIDE.  701 

These  weak  acids  are  thought  to  be  in  part  phosphoric  acid  and  in 
part  globuHns.  The  importance  of  the  alkaU  phosphates  for  the  carbon- 
dioxide  combination  has  been  shown  by  the  investigations  of  Ferxet; 
but  the  quantity  of  these  salts  in  the  semm  is.  at  least  in  certain  kinds 
of  blood,  for  example  in  ox-serum,  so  small  that  it  can  hardly  be  of 
importance.  In  regard  to  the  globulins  Setschexow  is  of  the  opinion 
that  they  do  not  act  as  acids  themselves,  but  form  a  combination  with 
carbon  dioxide,  producing  carboglobulinic  acid,  which  unites  with  the 
alkali.  According  to  Sertoli,^  whose  \'iews  have  foimd  a  supporter  in 
ToRUP.  the  globulins  themselves  are  the  acids  which  are  combined  with 
the  alkali  of  the  blood-serum.  In  both  cases  the  globulins  would  form, 
directly  or  indirectly,  that  chief  constituent  of  the  plasma  or  of  the  blood- 
senmi  which,  according  to  the  law  of  mass  action,  contends  with  the 
carbon  dioxide  for  the  alkalies.  By  a  greater  partial  pressure  of  the 
carbon  dioxide  the  latter  deprives  the  globulin  alkali  of  a  part  of  its  alkali 
and  bicarbonate  is  formed;  by  low  partial  pressure  carbon  dioxide  is  set 
free  and  it  is  abstracted  from  the  bicarbonate  by  the  globulin  alkali. 

The  assumption  that  the  proteins  of  the  blood  are  bodies  active  in 
combining  with  the  carbon  dioxide  has  received  some  support  by  the 
investigations  of  Siegfried  -  on  the  combination  of  carbon  dioxide  by 
amphoteric  amino  bodies.  Siegfried  has  found  that  amino  acids  com- 
bine   ■v^ith    carbon    dioxide,  therebv   being    converted    into  carbamino- 

H 

acids  (glycocoll)  for  example,  into  carbamino  acetic  acid  CH.^ — X — COOH 

I 

COOH 

and  that  the  carbon  dioxide  oan  be  readily  split  off  from  these  compounds. 
The  peptones  and  serum  proteids  in  the  presence  of  calcium  hydroxide 
may  also  act  in  the  same  manner  as  amino  acids.  Proteid  carbamino 
acids  are  formed,  and  the  possibility  of  such  a  binding  of  carbon  dioxide 
must  also  be  considered. 

In  the  foregoing  it  has  been  assumed  that  the  alkali  is  the  most  essen- 
tial and  important  constituent  of  the  blood-seriun,  as  well  as  of  the  blood 
m  general,  in  uniting  with  the  carbon  dioxide.  The  fact  that  the  quan- 
tity of  carbon  dioxide  in  the  blood  greatly  diminishes  with  a  decrease  in 
the  quantity  of  alkali  strengthens  this  assumption.  Such  a  condition 
is  found,  for  example,  after  poisoning  with  mineral  acids.  Thus  Walter 
found  only  2-.3  vols,  per  cent  carbon  dioxide  in  the  blood  of  rabbits  into 
whose  stomachs  h^'drochloric  acid  had  been  introduced.  In  the  coma- 
tose state  of  diabetes  mellitus  the  alkali  of  the  blood  seems  to  be  in  great 

*  Hoppe-Seyler.  Med.  chem.  Untersuch. 

*  Zeitschr.  f.  physiol.  Chem.,  44  and  46, 


702  CHEMISTRY  OF  RESPIRATION. 

part  saturated  with  acid  combinations,  /3-oxybutyric  acid  (Stadelmann, 
Minkowski),  and  Minkowski^  found  only  3.3  vols,  per  cent  carbon 
dioxide  in  the  blood  in  diabetic  coma. 

Gases  of  the  Lymph  and  Secretions. 

The  gases  of  the  lymph  are  the  same  as  in  the  blood-serum,  and  the 
lymph  stands  close  to  the  blood-serum  in  regard  to  the  quantity  of  the 
various  gases,  as  well  as  to  the  kind  of  carbon-dioxide  combination.  The 
investigations  of  Daenhardt  and  Hexsen  ^  on  the  gases  of  human  lymph 
are  at  hand,  but  it  still  remains  a  question  whether  the  lymph  investi- 
gated was  quite  normal.  The  gases  of  normal  dog-lymph  were  first  inves- 
tigated by  Hammarsten.'  These  gases  contained  traces  of  oxygen  and 
consisted  of  37.4-53.1  per  cent  CO.  and  1.6  per  cent  N  at  0°  C.  and  760 
mm.  Hg  pressure.  About  one  half  of  the  carbon  dioxide  w^as  in  firm 
chemical  combination.  The  quantity  was  greater  than  in  the  serum 
from  arterial  blood,  but  smaller  than  from  venous  blood. 

The  remarkable  observation  of  Buchxer  that  the  lymph  collected 
after  asphyxiation  is  poorer  in  carbon  dioxide  than  that  of  the  breathing 
animal  is  explained  by  Zuxtz  *  b}^  the  formation  of  acid  in  the  tissues, 
and  especially  in  the  lymphatic  glands,  immediately  after  death,  and  this 
acid  decomposes  the  alkali  carbonates  of  the  lymph  in  part. 

The  secretions  with  the  exception  of  the  saliva,  in  which  Pfltjger  and 
KuLZ  found  respectively  0.6  per  cent  and  1  per  cent  oxygen,  are  nearly 
free  from  oxygen.  The  quantity  of  nitrogen  is  the  same  as  in  blood,  and 
the  chief  mass  of  the  gases  consists  of  carbon  dioxide.  The  quantity  of 
this  gas  is  chiefly  dependent  upon  the  reaction,  i.e.,  upon  the  quantity  of 
alkali.  This  follows  from  the  analyses  of  Pfluger.  He  found  19  per 
cent  carbon  dioxide  removable  by  the  air-pump  and  54  per  cent  firmly 
combined  carbon  dioxide  in  a  strongly  alkaline  bile,  but,  on  the  contrary, 
6.6  per  cent  carbon  dioxide  removable  by  the  air-pump  and  0.8  per  cent 
firmly  combined  carbon  dioxide  in  a  neutral  bile.  Alkaline  saliva  is  also 
very  rich  in  carbon  dioxide.  As  average  for  two  analyses  made  by  Pflu- 
ger of  the  submaxillary  saliva  of  a  dog  we  have  27.5  per  cent  carbon 
dioxide  removable  by  the  air-pump  and  47.4  per  cent  chemically  com- 
bined carbon  dioxide,  making  a  total  of  74.9  per  cent.  KiJLz^  found  a 
maximum  of  65.78  per  cent  carbon  dioxide  for  the  parotid  saliva,  of 

1  Walter,  Arch.  f.  exp.  Path.  u.  Pharm.,  7;  Stadelmann,  ibid.,  17;  ^linkowski,  Jlittheil 
a.  d.  med.  Klink  in  Konigsberg,  1888. 

2  Virchow's  Arch.,  37. 

^  Ber.  d.  k.  sachs.  Gesellsch.  d.  Wissensch.,  math.-phys.  Kl.as.se,  23. 
*  Buchner,  Arbeiten  aus  der  pliysiol.  An.stalt  zii  Leipzig,  1876:  Zuntz,  1.  c,  85. 
^Pfluger,  Pfliiger's  Arch.,  1  and  2:  Kiilz.  2^itschr.  f.  Biologie,  23.     It  seems  as  if 
KiJlz's  results  were  not  calcidated  at  760  millimeters  Hg,  but  rather  at  1  meter. 


EXCHANGE  OF  GAS.  703 

which  3.31  per  cent  was  removable  by  the  air-pump  and  62.47  per  cent 
was  firmly  combined.  From  these  and  other  statements  on  the  quantity 
of  carbon  dioxide  removable  by  the  air-pump  and  chemically  combined 
in  the  alkaline  secretions  it  follows  that  bodies  occur  in  them,  although 
not  in  appreciable  quantities,  which  are  analogous  to  the  proteid  bodies 
of  the  blood-serum  and  which  act  like  weak  acids. 

The  acid  or  at  any  rate  non-alkaline  secretions,  urine  and  milk,  con- 
tain, on  the  contrary,  considerably  less  carbon  dioxide,  which  is  nearly 
all  removable  by  the  air-pump,  and  a  part  seems  to  be  loosely  combined 
with  the  sodium  phosphate.  The  figures  found  by  Pfluger  for  the 
total  quantity  of  carbon  dioxide  in  milk  and  urine  are  10  and  IS.  1-19.7 
per  cent  respectively. 

EwALD  ^  has  made  investigations  on  the  quantity  of  gas  in  pathological 
transudates.  He  found  only  traces,  or  at  least  only  very  insignificant 
quantities  of  oxgyen  in  these  fluids.  The  quantity  of  nitrogen  was  about 
the  same  as  in  blood;  that  of  carbon  dioxide  was  greater  than  in  the 
lymph  (of  dogs),  and  in  certain  cases  even  greater  than  in  the  blood  after 
asphyxiation  (dog's  blood).  The  tension  of  the  carbon  dioxide  was 
greater  than  in  venous  blood.  In  exudates  the  quantity  of  carbon  diox- 
ide, especially  that  firmly  combined,  increases  with  the  age  of  the  fluid 
while,  on  the  contrary,  the  total  quantity  of  carbon  dioxide,  and  espe- 
cially the  quantity  firmly  combined,  decreases  with  the  quantity  of  pus- 
corpuscles. 

II.  The  Exchange  of  Gas  between  the  Blood  on  the  One  Hand 
and  Pulmonary  Air  and  the  Tissues  on  the  other. 

In  the  introduction  (Chapter  I,  p.  3)  it  was  stated  that  we  are  to-day  of 
the  opinion,  derived  especially  from  the  researches  of  Pfluger  and  his 
pupils,  that  the  oxidations  of  the  animal  body  do  not  take  place  in  the 
fluids  and  juices,  but  are  connected  with  the  form-elements  and  tissues.  It 
is  nevertheless  true  that  oxidations  take  place  in  the  blood,  although  only 
to  a  slight  extent;  but  these  oxidations  depend,  it  seems,  upon  the  form- 
elements  of  the  blood,  hence  it  does  not  contradict  the  above  statement 
that  the  oxidations  occur  exclusively  in  the  cells  and  chiefly  in  the  tissues. 

The  gaseous  exchange  in  the  tissues,  which  has  been  designated  internal 
respiration,  consists  chiefly  in  that  the  oxygen  passes  from  the  blood  in  the 
capillaries  to  the  tissues,  while  the  great  bulk  of  the  carbon  dioxide  of  the 
tissues  originates  therein  and  passes  into  the  blood  of  the  capillaries.  The 
exchange  of  gas  in  the  lungs,  which  is  called  external  respiration,  consists, 
as  is  seen  by  a  comparison  of  the  inspired  and  expired  air,  in  the  blood 
taking  oxygen  from  the  air  in  the  lungs  and  giving  off  carbon  dioxide. 

'  C.  A.  Ewald,  Arch.  f.  (Anat.  u.)  Physiol.,  1873  and  1876. 


704  CHEMISTRY   OF  RESPIRATIOX. 

This  does  not  exclude  the  fact  that  in  the  lungs,  as  in  every  other  tissue,  an 
internal  respiration  takes  place,  namely,  a  combustion  with  a  consump- 
tion of  oxygen  and  formation  of  carbon  dioxide.  According  to  Bohr  and 
Henriques  ^  the  lungs  take  a  very  variable  but  ahvaj's  an  important 
part  in  the  total  metabolism.  This  part  which  on  an  average  is  33  per 
cent  but  may  even  rise  above  60  per  cent  of  the  total  metabolism  depends 
according  to  these  experimenters  upon  the  fact  that  the  intermediary  meta- 
bolic products  formed  in  the  tissue  are  burnt  in  the  lungs.  It  is  also  in 
part  represented  by  secretory  work  of  the  lungs. 

What  kind  of  processes  take  part  in  this  double  exchange  of  gas?  Is 
the  gaseous  exchange  simply  the  result  of  an  unequal  tension  of  the  blood 
on  one  side  and  the  air  in  the  lungs  or  tissues  on  the  other?  Do  the  gases 
pass  from  a  place  of  higher  pressure  to  one  of  a  lower,  according  to  the  laws 
of  diffusion,  or  are  other  forces  and  processes  active? 

These  questions  are  closely  related  to  that  of  the  tension  of  the  oxygen 
and  carbon  dioxide  in  the  blood  and  in  the  air  of  the  lungs  and  tissues. 

Oxygen  occurs  in  the  blood  in  a  disproportionately  large  part  as  oxy- 
hsemoglobin,  and  the  law  of  the  dissociation  of  oxyhsemoglobin  is  of  funda- 
mental importance  in  the  study  of  the  tension  of  the  oxygen  in  the  blood. 

Attempts  have  been  made  to  prove  this  law  by  investigations  on  a 
pure  solution  of  haemoglobin  and  HtJFXER  ^  has  made  very  careful  and 
important  determinations  on  such  solutions.  Recent  investigations  of 
Bohr  ^  and  his  pupils,  as  well  as  of  Loewy  and  Zuxtz,'*  have  shown  that 
the  conditions  in  the  blood  are  different  from  a  pure  haemoglobin  solution, 
which,  in  part,  may  be  due  to  a  change  in  the  hgemoglobin  brought  about 
in  its  preparation.  A  haemoglobin  solution  combines  firmer  with  oxygen 
than  the  blood,  and  the  dissociation  tension  of  the  oxygen  is  greater  in 
blood  than  in  a  haemoglobin  solution.  If  we  graphically  represent  the 
influence  of  the  oxygen  pressure  upon  the  power  of  the  blood  to  take  up 
oxygen  by  representing  the  oxygen  tension  as  abscissa  and  the  quantity 
of  oxygen  taken  up  as  ordinate  then  the  haemoglobin  solution  shows  a 
somewhat  flatter  oxygen  tension  curve  than  the  blood. 

The  oxygen  tension  may  be  variable,  as  Loewy  ^  has  shown,  with 
different  individuals  and,  as  Bohr,  Hasselbalch,  and  Krogh  «  have  found, 
that  besides  this  the  CO2  present  also  influences  the  oxygen  taken  up,  in 
that   as  the   carbon  dioxide  tension    (also  within  physiological   limits) 


»  Centralbl.  f.  Physiol.  6  and  Maly's  Jahresber,  27. 

=>  Arch.  f.  (Anat.'u.)  Physiol.,  1890  and  1894. 

3  See  Nagel's  Handbuch  and  Krogh,  Skand.,  Arch.  f.  Physiol.,  16. 

*Arch.  f.  (Anat.  u.)  Physiol.,  1904. 

^Ibid. 

•Centralbl.  f.  Physiol.  17  and  Skand.,  Arch.  f.  Physiol,  16. 


OXYGEN   TENSION   IN  THE   BLOOD.  705 

increases  the  oxygen  taken  up  diminishes.  The  laws  of  oxygen  absorption 
must  be  determined  by  determinations  upon  blood  itself  at  the  same  time 
observing  the  temperature  and  the  carbon  dioxide  tension.  A  series  of 
determinations  made  by  Krogh  ^  upon  horse's  blood  at  38°  and  a  constant 
carbon  dioxide  tension  will  be  given  below.  In  calculating  the  results  in 
column  5  the  quantity  of  oxygen  chemically  combined  at  150  mm.  oxygen 
pressure  is  equal  to  100. 

In  100  cc.  Blood  Oxygen  taken  up 

n,T.«^.,      Chemically       Oxygen  Per  cent  Tlic<;nlve.i  in 

10  6.0  0.020  30.0  0.030 

20  12.9  0.041  64.7  0.061 

30  16.3  0.061  81.6  0.091 

40  18.1  0.081  90.4  0.121 

50  19.1  0.101  95.4  0.152 

60  19.5  0.121  97.6  0.182 

70  19.8  0.141  98.8  0.212 

80  19.9  0.162  99.5  0.243 

90  19.95  0.182  99.8  0.273 

150  20.00  0.303  100.0  0.4.55 

From  the  above  table  we  see  that  even  with  an  oxygen  tension 
which  only  amounts  to  one  half  of  the  oxygen  pressure  in  the  air  that 
haemoglobin  in  greatest  part  is  saturated  with  oxygen.  The  dissociation 
is  hence  at  70-80  mm.  pressure  only  slightly  more  than  with  a  pressure  of 
150  mm.  and  indeed  even  with  as  low  a  pressure  as  40-30  mm.  still  90 
-80  per  cent  of  the  entire  quantity  of  oxygen  taken  up  chemically  at  150 
mm.  is  combined  with  the  haemoglobin. 

From  these  and  other  observations  it  follows  that  the  oxygen  partial 
pressure  may  sink  to  one  half  of  that  existing  in  the  atmospheric  air  without 
markedly  influencing  the  oxygen  content  of  the  blood.  This  coincides 
also  with  the  experience  of  Frankel  and  Geppert  -  on  the  action  of  low 
air  pressures  upon  the  oxygen  content  of  the  blood  of  dogs.  With  an  air 
pressure  of  410  mm.  Hg  they  found  that  the  oxygen  content  of  arterial 
blood  was  normal.  With  an  air  pressure  of  378-365  mm.  it  was  slightly 
diminished  and  only  on  reducing  the  pressure  to  300  mm.  was  a  mention- 
able  decrease  observed.  A.  Loewy  ^  has  found  that  the  lowest  oxvgen 
pressure  of  the  alveolar  air  when  the  exchange  of  material  can  go  on 
normally  both  qualitatively  and  quantitatively,  is  equal  to  30  mm.  Hg. 

It  may  be  concluded  from  the  large  quantity  of  oxygen  or  oxvhaemo- 
globin  in  the  arterial  blood  that  the  tension  of  the  oxygen  in  the  arterial 
blood   must   be    relatively  higher.     From    the    investigations    of   several 

1  Skand.  Arch,  f.  Physiol.,  16. 

^  Uber  die  Wirknugen  der  verdriinnten  Luft  auf.  den.  Orsanismus.  Berlin,  1883. 
*A.  Loewy,  L^ntersuch.,  iiber  die  Respiration  und  Zirculation  etc.,  Berlin  1895;  also 
Centralbl.  f.  Physiol.,  13,  449  and  Arch.  f.  (.Inat.  u.)  Physiol,  1900. 


706  CHEMISTRY  OF  RESPIRATION. 

experimenters,  such  as  P.  Bert,  Herter,  and  Hufxer,^  who  experi- 
mented partly  on  living  animals  and  partly  with  hsemoglobin  solutions, 
we  may  assume  the  tension  of  the  oxygen  in  arterial  blood  at  the  tem- 
perature of  the  body  to  be  equal  to  a  partial  oxygen  pressure  of  75-80 
mm.  Hg. 

According  to  Bohr  -  the  facts  are  otherwise,  and  he  has  obtained 
remarkably  higher  results  for  the  oxygen  tension  in  arterial  blood. 

He  experimented  on  dogs  allowing  the  blood,  whose  coagulation  had  been 
prevented  by  the  injection  of  peptone  solution  or  infusion  of  the  leech,  to  flow 
from  one  bisected  carotid  to  the  other,  or  from  the  femoral  artery  to  the  femoral 
vein,  through  an  apparatus  called  by  him  an  haemataerometer.  The  apparatus, 
which  is  a  modification  of  Ludwig's  rheometer  {stromuhr),  allowed,  according 
to  Bohr,  of  a  complete  interchange  between  the  gases  of  the  blood  circulating 
through  the  apparatus  and  a  quantity  of  gas  whose  composition  was  known  at 
the  beginning  of  the  experiment  and  enclosed  in  the  apparatus.  The  mixture 
of  gases  was  analyzed  after  an  equalization  of  the  gases  by  diffusion.  In  this 
way  the  tension  of  the  oxygen  and  carbon  dioxide  in  the  circulating  arterial  blood 
was  determined.  During  the  experiment  the  composition  of  the  inspired  and 
expired  air  was  also  determined,  the  number  of  inspirations  noted,  and  the  extent 
of  respiratory  exchange  of  gas  measured.  To  be  able  to  make  a  comparison 
between  the  gas  tension  in  the  blood  and  in  an  expired  air  whose  composition  was 
closer  to  the  unknown  composition  of  the  alveolar  air  than  the  ordinary  expired 
air,  the  composition  of  the  expired  air  at  the  moment  it  passed  the  bifurcation  of 
the  trachea  was  ascertained  by  special  calculation.  The  ten.sion  of  the  gases  in 
this  "bifurcated  air"  could  be  compared  with  the  tension  of  the  gases  of  the  blood, 
and  in  such  a  way  that  the  comparison  took  place  simultaneously. 

Bohr  found  remarkably  high  results  for  the  oxygen  tension  in  arterial 
blood  in  this  series  of  experiments.  They  varied  between  101  and  144 
mm.  Hg  pressure.  In  eight  out  of  nine  experiments  on  the  breathing  of 
atmospheric  air.  and  in  four  out  of  five  experiments  on  breathing  air  con- 
taining carbon  dioxide,  the  oxygen  tension  in  the  arterial  blood  was  higher 
than  the  "bifurcated  air."  The  greatest  difference,  where  the  oxygen 
tension  was  higher  in  the  blood  than  in  the  air  of  the  lungs,  was  38  mm.  Hg. 

HiJFXER  and  Fredericq^  have  made  the  objection  to  Bohr's  experi- 
ments and  views  that  a  perfect  equilibrium  had  probably  not  been  attained 
between  the  air  in  the  apparatus  and  the  gases  of  the  blood.  Fredericq. 
by  new  experiments,  has  presented  strong  objections  to  the  acceptance  of 
Bohr's  findings,  while  on  the  other*  hand  Bohr  not  only  defends  his  experi- 
ments but  also  finds  errors  in  the  experiments  of  his  opponents.  On  the 
other  hand  Hald.vxe  and  Smith's  *  experiments  making  use  of  an  entirel}^ 
different  principle  speak  for  the  high  results  found  by  Bohr. 

'  Bert,  La  pre.s.sion  barometrique,  Paris,  1878;  Herter,  Zeitschr.  f.  physiol.  Chem., 
3:  Hiifner,  1.  c. 

=  Skand.  Arch.  f.  Physiol.  2  and  Xagel's  Handbuch.  der  Physiologie. 

3  Hiifner,  Arch.  f.  (.\nat.  u.)  Physiol.  1S90;  FreJericq,  Centralbl.  f.  Physiol.  7  and 
Traveanx  du  laboratoire  de  I'lnstitiite  de  physiologie  de  Li^ge  5.  1S96. 

*Haldane,  Journ.  of  Phj'siol.,  18;  Haldane  and  Smith,  ibid.,  20. 


COMPOSITION   OF  THE   ALVEOLAR  AIR.  707 

Haldane's  method  is  as  follows:  The  individual  experimented  upon  is  allowed 
to  inspire  air  containing  an  exactly  known  but  small  quantity  of  carbon  monoxide 
(0.045-0.06  per  cent),  until  no  further  absorption  of  carbon  monoxide  takes  place 
and  the  percentage  saturation  of  the  haemoglobin  in  the  arterial  blood  with  carbon 
monoxide  has  become  constant,  as  shown  by  a  special  titration  method.  This 
percentage  saturation  is  dependent  upon  the  relation  between  the  tension  of  the 
oxygen  in  the  blood  and  the  tension  of  the  carbon  monoxide,  as  known  from  the 
composition  of  the  inspired  air.  When  this  last  and  the  percentage  saturation 
with  carbon  monoxide  and  oxygen  are  known  the  oxygen  tension  in  the  blood  can 
be  easily  calculated. 

According  to  this  method  Haldane  and  Smith  found  still  higher 
figures  than  Bohr  for  the  oxygen  tension  in  the  blood,  and  they  calculated 
the  average  tension  of  the  oxygen  in  human  arterial  blood  as  38.5  per  cent 
of  an  atmosphere  i.e.,  equal  to  about  293  mm.  Hg. 

Let  us  now  compare  the  figures  for  the  oxygen  tension  of  the  arterial 
blood  as  found  by  various  investigators  with  the  tension  of  the  oxygen  in 
the  air  of  the  lungs. 

Numerous  investigations  as  to  the  composition  of  the  inspired  atmos- 
pheric air  as  well  as  the  expired  air  are  at  hand,  and  it  can  be  said  that 
these  two  kinds  of  air  at  0°  C.  and  a  pressure  of  760  mm.  Hg  have  the  fol- 
lowing average  composition  in  volume  per  cent: 

Oxygen. 

Atmospheric  air 20.96 

Expired  air 16.03 

The  partial  pressure  of  the  oxygen  of  the  atmospheric  air  corresponds 
at  a  normal  barometric  pressure  of  760  mm.  to  a  pressure  of  160  mm.  Hg. 
The  loss  of  oxygen  which  the  inspired  air  suffers  in  respiration  amounts  to 
about  4.93  per  cent,  while  the  expired  air  contains  about  one  hundred 
times  as  much  carbon  dioxide  as  the  inspired  air. 

The  expired  air  is  therefore  a  mixture  of  alveolar  air  with  the  residue 
of  inspired  air  remaining  in  the  air-passages;  hence  in  the  study  of  the 
gaseous  exchange  in  the  lungs  the  alveolar  air  must  first  be  considered. 
There  does  not  exist  any  direct  determination  of  the  composition  of  the 
alveolar  air  in  man,  but  only  approximate  calculations.  From  the  average 
results  found  by  Vierordt  in  normal  respiration  for  the  carbon  dioxide 
in  the  expired  air,  4.63  per  cent,  Zuntz  ^  has  calculated  the  probable 
quantity  of  carbon  dioxide  in  the  alveolar  air  as  equal  to  5.44  per  cent. 
If  we  start  from  this  value,  with  the  assumption  that  the  quantity  of  nitro- 
gen in  the  alveolar  air  does  not  essentially  differ  from  the  expired  air,  and 
admit  that  the  quantity  of  oxygen  in  the  alveolar  air  is  6  per  cent  less 
than  the  inspired  air,  it  will  be  seen  that  the  alveolar  air  contains  15  per 
cent  oxygen.     As  the  total  pressure  of  the  air  of  the  lungs  after  deducting 


Nitrogen 
(and  argon). 

Carbon 
Dioxide. 

79.02 

0.03 

79.59 

4.38 

'  See  Zuntz  I.  c.  Hennann's  Handbuch  105  and  106. 


708  CHEMISTRY  OF  RESPIRATION. 

the  aqueous  tension  of  about  50  mm.  can  be  calculated  as  about  710  mm. 
the  partial  pressure  of  the  oxygen  in  man  can  be  put  at  about  106  mm, 
and  that  of  the  carbon  dioxide  as  about  45  mm. 

There  are  several  direct  determinations  of  the  alveolar  air  of  dogs  by 
Pfluger  and  his  pupils  Wolffberg  and  Nussbaum.^  These  determina- 
tions which  show  that  the  alveolar  air  is  not  much  richer  in  carbon  dioxide 
than  the  expired  air  have  been  performed  by  means  of  the  so-called  lung- 
catheter. 

The  principle  of  this  method  is  as  follows:  By  the  introduction  of  a  catheter 
of  a  special  construction  into  a  branch  of  a  bronchus  the  corresponding  lobe  of 
the  lung  may  be  hermetically  sealed,  while  in  the  other  lobes  of  the  same  lung,  and 
in  the  other  lung,  the  ventilation  remains  unchanged,  so  that  no  accumulation 
of  carbon  dioxide  takes  place  in  the  blood.  When  the  cutting  off  lasts  so  long  that 
a  complete  equalization  between  the  gases  of  the  blood  and  the  retained  air  of 
the  lungs  is  assumed,  a  sample  of  this  air  of  the  lungs  is  removed  by  means  of 
the  catheter  and  analyzed. 

In  the  air  thus  obtained  from  the  lungs  Wolffberg  and  Nussbaum 
found  an  average  of  3.6  per  cent  COj.  Nussbaum  has  also  determined  the 
carbon-dioxide  tension  in  the  blood  from  the  right  heart  in  a  case  simul- 
taneous with  the  catheterization  of  the  lungs.  He  found  nearly  identical 
results,  namely,  a  carbon-dioxide  tension  of  3.84  per  cent  and  3.81  per  cent 
of  an  atmosphere,  which  also  shows  that  complete  equalization  between 
the  gases  of  the  blood  and  lungs  in  the  enclosed  parts  of  the  lungs  had 
taken  place.  From  these  investigations  it  can  be  calculated  that  the 
quantity  of  oxygen  in  the  alveolar  air  of  dogs  is  about  16  per  cent,  which 
corresponds  to  an  oxygen  partial  pressure  of  about  115  mm.  Hg. 

If  the  oxygen  partial  pressure  in  the  alveoUi,  is  put  at  only  106-115  mm. 
Hg,  and  compare  this  with  about  80  mm.  as  found  by  certain  investigators 
for  the  oxygen  tension  of  the  arterial  blood,  we  find  that  a  considerable 
excess  remains  in  favor  of  the  alveolii,  and  the  taking  up  of  oxygen  in  the 
lungs  can  simply,  according  to  physical  laws,  be  explained  as  a  diffusion 
process.  The  conditions  are  quite  different  if  we  start  with  the  high- 
tension  results  of  Bohr,  101-144  mm.  Hg,  or  the  still  higher  results  of 
Haldane  and  Smith.  The  oxygen  tension  in  the  blood  is  in  many  cases, 
according  to  Haldane  and  Smith,  as  average  for  various  races  of  animals, 
indeed  always  higher  than  the  tension  in  the  lungs.  In  these  cases  the 
passage  of  oxygen  from  the  lungs  to  the  blood  cannot  be  simply  explained 
by  a  diffusion.  We  must  therefore,  with  Bohr,  accept  a  special  specific 
activity  of  the  lungs,  and  according  to  him  a  secretory  activity  of  the 
lungs  also  exists  besides  diffusion. 

As  the  views  on  the  taking  up  of  oxygen  are  disputed  so  also  are  the 
views  on  the  giving  up  of  carbon  dioxide. 

'  Wolffberg,  Pfliiger's  Arch.  6;  Nussbaum,  ibul.  7. 


CARBON   DIOXIDE  TENSION.  709 

The  tension  of  the  carbon  dioxide  in  the  blood  has  been  determined  in 
different  ways  by  Pflijger  and  his  pupils,  Wolffberg,  Strassburg,  and 

NUSSBAUM.^ 

According  to  the  aerotonometric  method  the  blood  is  allowed  to  flow  directly 
from  the  artery  or  vein  through  a  glass  tube  which  contains  a  gas  mixture  of  a 
known  composition.  If  the  tension  of  the  carbon  dioxide  in  the  blood  is  greater 
than  the  gas  mixture,  then  the  blood  gives  up  carbon  dioxide,  while  in  the  reverse 
case  it  takes  up  carbon  dioxide  from  the  gas  mixture.  The  analysis  of  the  gas 
mixture  after  passing  the  blood  through  it  will  also  decide  if  the  tension  of  the 
carbon  dioxide  in  the  blood  is  greater  or  less  than  in  the  gas  mixture;  and  by  a 
sufficiently  great  number  of  determinations,  especially  when  the  cjuantity  of  carbon 
dioxide  of  the  gas  mixture  corresponds  as  nearly  as  possible  in  the  beginning  to 
the  probable  tension  of  this  gas  in  the  blood,  we  may  learn  the  tension  of  the 
carbon  dioxide  in  the  blood. 

According  to  this  method  the  carbon-dioxide  tension  of  the  arterial 
blood  is  on  an  average  2.8  per  cent  of  an  atmosphere,  corresponding  to  a 
pressure  of  21  mm.  mercury  (Strassburg).  In  the  blood  from  the  pul- 
monary alveoli  Nussbaum  found  a  carbon-dioxide  tension  of  3.81  per  cent 
of  an  atmosphere,  corresponding  to  a  pressure  of  28.95  mm.  mercury. 
Strassburg,  who  experimented  in  non-tracheotomized  dogs  in  which  the 
ventilation  of  the  lungs  was  less  active  and  therefore  the  carbon  dioxide 
was  removed  from  the  blood  with  less  readiness,  found  in  the  venous  blood 
of  the  heart  a  carbon-dioxide  tension  of  5.4  per  cent  of  an  atmosphere, 
corresponding  to  a  partial  pressure  of  41.01  mm.  mercury. 

Another  method  is  the  catheterization  of  a  lobe  of  the  lungs  (see  page 
708).  In  the  air  thus  obtained  from  the  lungs  Nussbaum  and  Wolffberg 
found  an  average  of  3.6  per  cent  CO2.  Nussbaum,  as  previously  mentioned, 
has  also  determined  the  carbon-dioxide  tension  in  the  blood  of  the  pul- 
monary alveoli  in  a  case  simultaneously  with  the  catheterization  of  the 
lungs.  He  found  nearly  identical  results,  namely,  a  carbon-dioxide 
tension  of  3.84  per  cent  and  3.81  per  cent. 

According  to  these  investigations  the  giving  up  of  carbon  dioxide  may 
also  be  explained  by  physical  laws;  but  Bohr,  in  his  experiments  above 
mentioned  (page  706),  has  arrived  at  other  results  in  regard  to  the  carbon- 
dioxide  tension.  In  eleven  experiments  with  inhalation  of  atmospheric 
air  the  carbon-dioxide  tension  in  the  arterial  blood  varied  from  0  to  38 
mm.  Hg,  and  in  five  experiments  with  inhalation  of  air  containing  carbon 
dioxide,  from  0.9  to  57.8  mm.  Hg.  A  comparison  of  the  carbon-dioxide 
tension  in  the  blood  with  the  bifurcated  air  gave  in  several  cases  a  greater 
carbon-dioxide  pressure  in  the  air  of  the  lungs  than  in  the  blood,  and  as 
maximum  this  difference  amounted  to  17.2  mm.  in  favor  of  the  air  of  the 
lungs  in  the  experiments  with  inhalation  of  atmospheric  air.  As  the 
alveolar  air  is  richer  in  carbon  dioxide  than  the  bifurcated  nir  this  experi- 

*  Wolffberg,  Pfliiger's  Arch.,  6;  Strassburg,  ibid.;  Nussbaum,  ibid.,  7. 


710  CHEMISTRY  OF  RESPIRATION. 

ment  unquestionably  proves,  according  to  Bohr,  that  the  carbon  dioxide 
has  migrated  against  the  high  pressure. 

In  opposition  to  these  investigations,  Fredericq,^  in  his  above-men- 
tioned experiments,  obtained  the  same  figures  for  the  carbon-dioxide  ten- 
sion in  arterial  peptone  blood  as  Pflltger  and  his  pupils  found  for  normal 
blood.  Weisgerber,^  in  Fredericq's  laboratory,  has  made  experiments 
with  animals  which  respired  air  rich  in  carbon  dioxide,  and  these  experi- 
ments confirm  Pfltjger's  theory  of  respiration.  Recently  Falloise  has 
made  determinations  of  the  carbon-dioxide  tension  of  venous  blood  by 
means  of  Fredericq's  aerotonometer.  The  carbon-dioxide  tension  was 
found  to  equal  6  per  cent  of  an  atmosphere,  hence  somewhat  higher  than 
the  results  found  by  PFLiJGER's  pupils.  In  opposition  to  these  investiga- 
tions Bohr  has  presented  strong  objections;  he  has  demonstrated  the 
principles  for  the  construction  of  the  tonometer  and  according  to  him  the 
older  experiments  with  the  tonometer  are  not  conclusive  as  he  claims  that 
a  complete  equilibrium  of  the  gas  tension  was  not  sufficiently  accomplished. 

A  certain  importance  has  been  ascribed  to  oxygen  in  regard  to  the 
elimination  of  carbon  dioxide  in  the  lungs,  in  that  it  has  an  expelling 
action  on  the  carbon  dioxide  from  its  combinations  in  the  blood.  This 
statement,  first  made  by  Holmgren,  has  recently  found  an  advocate 
in  Werigo.  Still  Zuntz  has  presented  very  important  objections  to 
Werigo's  experiments,  and  Bohr^  has  later  also  shown  that  we  have  no 
positive  basis  for  the  above  assumption. 

The  conditions  as  to  the  elimination  of  carbon  dioxide  in  the  lungs  is 
also  not  quite  clear,  and  from  the  above  we  see  that  in  regard  to  the  gas 
exchange  in  the  lungs  w^e  have  two  opposed  views.  According  to  the 
older  view  suggested  by  the  Pfluger  school  the  exchange  of  gas  follows 
the  simple  physical  laws  and  is  on  the  whole  a  diffusion  process.  Accord- 
ing to  Bohr's  view  a  diffusion  does  take  place;  but  according  to  him  the 
lung  is  a  gland  which  has  the  power  of  secreting  gases,  and  the  gas  exchange 
in  the  lungs  is  essentially  a  secretory  process.  According  to  Hammarsten 
we  cannot  dispute  the  fact  that  the  investigations  made  thus  far  speak 
very  much  in  favor  of  Bohr's  view,  and  this  latter  also  receives  support 
in  the  detectable  secretion  of  gases  in  certain  animals. 

That  a  true  secretion  of  gases  occurs  in  animals  follows  from  the  composition 
and  behavior  of  the  gases  in  the  swimming-bladder  of  fishes.  These  gases  con- 
sist of  oxvgen  and  nitrogen  with  only  small  quantities  of  carbon  dioxide.  In 
fishes  which  do  not  live  at  any  great  depth  the  quantity  of  oxygen  is  ordinarily 
as  high  as  in  the  atmosphere,  while  fishes  which  live  at  great  depths  may,  accord- 


'  See  footnote  3  page  706. 

'Centralbl.  f.  Physiol.  10,  482;  Falloise,  see  Maly's  Jahresber,  32. 
^Holmgren.  Wien  Sitzungsber.,  48  Werigo,  Pfluger's   Arch.,  51  and  52;  Zuntz,  ibul., 
52;  Bohn,  see  Nagel's  Handbuch  der  Physiologie. 


INTERNAL  RESPIRATION.  711 

ing  to  BiOT  and  others,  contain  considerably  more  oxygen  and  even  above  80  per 
cent.  MoREAU  has  also  found  that  after  emptying  the  swimming-bladder  by 
means  of  a  trocar  new  air  collected  after  a  time,  and  this  air  was  richer  in  oxygen 
than  the  atmospheric  air  and  contained  even  85  per  cent  oxygen.  Bohr,  who 
has  proved  and  confirmed  these  statements,  also  found  that  this  collection  is  under 
the  influence  of  the  nervous  system,  because  on  the  section  of  certain  branches 
of  the  pneumogastric  nerve  it  is  discontinued.  It  is  beyond  dispute  that  there  is 
here  a  secretion  and  not  a  diffusion  of  oxygen.  Recently  Jaeger  '■  has  given  a 
further  explanation  as  to  the  secretory  activity  of  the  swimming-bladder. 

From  what  has  been  said  above  (page  703)  in  regard  to  the  internal 
respiration,  one  can  conclude  that  it  consists  chiefly  in  that  in  the  capil- 
laries the  oxygen  passes  from  the  blood  into  the  tissues,  while  the  carbon 
dioxide  passes  from  the  tissues  into  the  blood. 

The  assertion  of  Estor  and  Saint  Pierre  that  the  quantity  of  oxygen 
in  the  blood  of  the  arteries  decreases  with  the  remoteness  from  the  heart 
has  been  shown  to  be  incorrect  by  Peluger,^  and  the  oxygen  tension  in  the 
blood  on  entering  the  capillaries  must  be  higher.  The  oxygen  tension  of 
the  plasma  is  of  importance  for  the  giving  up  of  oxygen  to  the  tissues  as 
the  blood  corpuscles  only  contain  a  supply  of  oxygen,  which,  as  the  tissue 
removes  oxygen  from  the  plasma,  replaces  this  again.  This  quantity  of 
oxygen  which  is  dissolved  in  the  plasma  and  at  the  disposal  of  the  tissues 
is  dependent  upon  the  oxygen  tension  in  the  blood  and  only  indirectly 
dependent  upon  the  total  quantity  of  oxygen  in  the  blood.  As  this  tissue 
is  nearly  or  entirely  free  from  oxygen  a  considerable  difference  in  regard 
to-  the  oxygen  pressure  must  exist  between  the  blood  and  the  tissues. 
The  possibility  that  this  difference  in  pressure  is  sufficient  to  supply  the 
tissues  with  the  necessary  quantity  of  oxygen  is  hardly  to  be  doubted. 

The  animal  body  it  seems  also  has  the  command  over  means  of  regu- 
lating and  varying  the  oxygen  tension,  and  such  a  means  is  the  carbon 
dioxide  produced  in  the  tissue  which,  according  to  Bohr,  Hasselbach, 
and  Krogh,^  raises  the  oxygen  tension.  Another  regulating  moment  is, 
according  to  Bohr,  the  specific  oxygen  capacity  of  the  blood  which  means 
the  relationship  of  the  maximum  oxygen  combination  to  the  quantity  of 
iron  of  the  blood  or  the  haemoglobin  solution. 

As  the  haemoglobin  obtained  from  different  blood  portions  does  not,  according 
to  Bohr,  always  take  up  the  same  quantity  of  oxygen  for  each  gram,  so  the 
haemoglobin  within  the  blood-corpuscle  may  show  a  similar  behavior.  He  calls 
the  quantity  of  oxygen  (measured  at  0°  C.  and  760  mm.  Hg  which  is  taken  up 
by  1  gram  of  haemoglobin  of  the  blood  at  15°  C.  and  an  oxygen  pressure  of  150  mm. 
the  specific  oxygen  capacity.*     This  c;[uantity,  he  claims,  may  be  different  not  only 

'  Biot,  see  Hermann's  Handbuch  d.  Physiol.,  4,  Thl.  2,  151 ;  Moreau,  Compt.  rend., 
57;  Bohr,  Journ.  of  Physiol.,  15.  See  also  Hiifner,  Arch.  f.  (Anat.  u.)  Physiol.,  1892; 
Jaeger,  Pfliiger's  Arch.,  94. 

^  Estor  and  Saint  Pierre  with  Pfliiger  in  Pfliiger's  Arch.  1. 

» L.  c. 

*Centralbl.  f.  Physiol.  4  and  Nagel's  Handbuch. 


712  CHEMISTRY  OF  RESPIRATION. 

in  different  individuals,  but  also  in  the  different  vascular  systems  of  the  same 
animal,  and  it  may  also  be  changed  experimentally  by  bleeding,  breathing  air 
deficient  in  oxygen,  or  poisoning.  It  is  now  evident  that  one  and  the  same  quan- 
tity of  oxygen  in  the  blood,  other  things  being  ecjual,  must  have  a  different  ten- 
sion according  as  the  specific  oxygen  capacity  is  greater  or  smaller.  The  tension 
of  the  oxygen,  Bohr  says,  may  be  changed  without  changing  the  quantity  of 
ox3rgen,  and  the  animal  body  must,  according  to  him,  have  means  of  varying  the 
tension  of  the  oxygen  in  the  tissues  in  a  short  time  without  changing  the  quantity 
of  oxygen  contained  in  the  blood.  The  great  importance  of  such  a  property  of 
the  tissues  for  respiration  is  evident;  but  it  is  perhaps  too  early  to  give  a  positive 
opinion  on  Bohr's  statements  and  experiments. 

In  regard  to  the  carbon-dioxide  tension  in  the  tissue  it  must  be  assumed 
a  priori  that  it  is  higher  than  in  the  blood.  This  is  found  to  be  true. 
Strassburg  ^  found  in  the  urine  of  dogs  and  in  the  bile  a  carbon-dioxide 
tension  of  9  per  cent  and  7  per  cent  of  an  atmosphere,  respectively.  The 
same  experimenter  has,  further,  injected  atmospheric  air  into  a  ligatured 
portion  of  the  intestine  of  a  living  dog  and  analyzed  the  air  taken  out  after 
some  time.  He  found  a  carbon-dioxide  tension  of  7.7  per  cent  of  an  atmos- 
sphere.  The  carbon-dioxide  tension  in  the  tissues  is  considerably  greater 
than  in  the  venous  blood,  and  there  is  no  opposition  to  the  view  that  the 
carbon  dioxide  simply  diffuses  from  the  tissues  into  the  blood  according  to 
the  laws  of  diffusion. 

Several  methods  have  been  suggested  f  )r  the  study  of  the  quantitative 
relationship  of  the  respiratory  exchange  of  gas.  The  reader  must  be 
referred  to  other  text-books  for  more  details  as  to  these  methods,  and  we 
will  here  only  mention  the  chief  features  of  the  most  important  methods. 

Regnault  and  Reiset's  Method.  According  to  this  method  the  animal  or 
person  experimented  upon  is  allowed  to  respire  in  an  enclosed  space.  The  carbon 
dioxide  is  removed  from  the  air,  as  it  forms,  by  strong  caustic  alkali,  from  which 
the  quantity  may  be  determined,  while  the  oxygen  is  replaced  continually  by 
exactly  measured  quantities.  This  method,  which  also  makes  possible  a  direct 
determination  of  the  oxygen  used  as  well  as  the  carbon  dioxide  produced,  has  since 
been  modified  by  other  investigators,  such  as  Pfluger  and  his  pupils,  Seegen 
and  XowAK,  and  Hoppe-Sevler,  Rosenthal,  and  Zuntz.^ 

Pettenkofer's  Method.  According  to  this  method  the  individual  to  be 
experimented  upon  breathes  in  a  room  through  which  a  current  of  atmospheric 
air  is  passed.  The  quantity  of  air  passed  through  is  carefully  measured.  As  it 
is  impossible  to  analyze  all  the  air  made  to  pass  through  the  chamber,  a  small 
fraction  of  this  air  is  diverted  into  a  branch  line  during  the  entire  experiment, 
carefully  measured,  and  the  quantity  of  carbon  dioxide  and  water  determined. 
From  the  composition  of  this  air  the  quantity  of  water  and  carbon  doxide  con- 
tained in  the  large  quantity  of  air  made  to  pass  through  the  chamber  can  be 
calculated.  The  consumption  of  oxygen  cannot  be  directly  determined  in  this 
method,  but  may  be  calculated  indirectly  by  difference,  which  is  a  defect  in  this 

'  Pfliiger's  Arch.  6. 

^  See  Zuntz  in  Hermann's  Handbuch,  4,  Thl.  2,  and  Hoppe-Seyler,  Zeitschr.  f . 
physiol.  Chem.,  19;  Rosenthal,  Arch.  f.  (Anat.  u.)  Phj^siol.,  1902;  Zuntz,  Verhandl.  d. 
Berl.  physiol.  Gesellsch.,  1901. 


LUNGS   AND  THEIR  EXPECTORATIONS.  713 

method.  The  large  respiration  apparatus  of  Sonden  and  Tigepstedt  as  well  as 
of  AtwatePv  and  Rosa  ^  are  based  upon  this  principle. 

Speck's  Method.-  For  briefer  experiments  on  maa  .'Speck  has  used  the  follow- 
ing: He  breathes  into  two  spirometer-reeeivers,  on  which  the  gas- volume  can  be 
read  off  very  accurately,  through  a  mouthpiece  with  two  valves,  closing  the  nose 
with  a  clamp.  The  air  from  one  of  the  spirometers  is  inhaled  through  one  valve 
and  the  expired  air  passes  through  the  other  into  the  other  spirometer.  By  means 
of  a  rubber  tube  connected  with  the  expiration-tube  an  accurately  measured  part 
of  the  expired  air  may  be  passed  into  an  absorption-tube  and  analyzed. 

ZuNTZ  and  Geppert's  Method.^  This  method,  which  has  been  improved  by 
ZuNTZ  and  his  pupils  from  time  to  time,  consists  in  the  following:  The  individual 
being  experimented  upon  inspires  pure  atmospheric  air  through  a  very  wide  feed- 
pipe leading  from  the  open  air,  the  inspired  and  the  expired  air  being  separated  by 
two  valves  (human  subjects  breathe  with  closed  nose  by  means  of  a  soft-rubber 
mouthpiece,  animals  through  an  air-tight  tracheal  canula).  The  volume  of  the 
expired  air  is  measured  by  a  gas-meter  and  an  aliquot  part  of  this  air  collected  and 
the  fiuantity  of  carbon  dioxide  and  oxygen  determined.  As  the  composition  of  the 
atmospheric  air  can  be  considered  as  constant  within  a  certain  limit,  the  production 
of  carbon  dioxide  as  well  as  the  consumption  of  oxygen  may  be  readily  calculated 
(see  the  works  of  Zuxtz  and  his  pupils). 

IIaxriot  and  Richet's  Method  *  is  characterized  by  its  simplicity.  These 
investigators  allow  the  total  air  to  pass  through  three  gasometers,  one  after  the 
other.  The  first  measures  the  inspired  air,  whose  composition  is  known.  The 
second  gasometer  measures  the  expired  air,  and  the  third  the  quantity  of  the 
expired  air  after  the  carbon  dio.xide  has  been  removed  by  a  suitable  apparatus. 
The  quantity  of  carbon  dioxide  produced  and  the  oxygen  consumed  can  be  readily 
calculated  from  these  data. 


APPENDIX. 

The  Lungs  and  their  E.xpectorations. 

Besides  proteid  bodies  and  the  albuminoids  of  the  connective-substance 
group,  lecithin,  taurine  (especially  in  ox-lungs),  i^ric  acid,  and  inosite  have 
been  found  in  the  lungs.  Poulet  ^  claims  to  have  found  a  special  acid, 
which  he  has  called  pulniotartaric  acid,  in  the  lung-tissue.  Glycogen 
occurs  abundantly  in  the  embryonic  lung,  but  is  absent  in  the  adult  organ. 
The  proteolytic  enzymes  also  belong  to  the  ph3'siological  constituents  of 
the  lungs.  They  are  active  in  the  autolysis  of  the  lungs  (Jacoby)  as  well 
as  in  the  solution  of  pneumonic  infiltrations  (Fr.  Muller^). 

^  Pettenkofer's  method;  see  Zuntz,  1.  c;  Sonden  and  Tigerstedt,  Skand.  Arch.  f. 
Physiol.,  6;  Atwater  and  Rosa,  Bull,  of  Dept.  of  Agriculture,  63.     Washington. 

^  Speck,  Physiologie  des  menschlichen  Atmens.     Leipzig,  1892. 

^  Pfliiger's  Arch.,  42.  See  also  Magnus-Levyin  Pfliiger's  Arch.,  55,  10,  in  which  the 
work  of  Zuntz  and  his  pupils  is  cited. 

*  Compt.  rend.,  104. 

5  Cited  from  Maly's  Jahresber.,  18,  248. 

'Jacoby,  Zeitschr.  f.  physiol.  Chem.,  33;  ^liiller,  Verhandl.  d.  Kongress.  f.  inn. 
Medizin,  1902. 


714  CHEMISTRY   OF   RESPIRATION. 

The  black  or  dark-brown  pigment  in  the  lungs  of  human  beings  and  domestic 
animals  consists  chiefly  of  carbon,  which  originates  from  the  soot  in  the  air.  The 
pigment  may  in  part  also  consist  of  melanin.  Besides  carbon,  other  bodies,  such 
as  iron  oxide,  silicic  acid,  and  clay,  may  be  deposited  in  the  lungs,  being  inhaled 
as  dust. 

Among  the  bodies  found  in  the  lungs  under  pathological  conditions 
must  be  specially  mentioned  proteoses  (and  peptones?)  in  pneumonia  and 
suppuration,  glycogen,  a  slightly  dextrorotatory  carbohydrate  differing 
from  glycogen  found  by  Pouchet  in  consumptives,  and  finally  also  cellu- 
lose, which,  according  to  Freund,^  occurs  in  the  lungs,  blood,  and  pus  of 
persons  with  tuberculosis. 

C.  W.  Schmidt  found  in  1000  grams  of  mineral  bodies  from  the  normal 
human  lu.ig  the  following:  NaCl  130,  K^O  13,  Na^O  195,  CaO  19,  MgO  19, 
Fe203  32,  P.O.,  485,  SOg  8,  and  sand  134  grams.  According  to  Oidtmann  ^ 
the  lungs  of  a  14-day  old  child  contained  793.05  p.  m.  water,  198.19  p.  m. 
organic  bodies,  and  5.76  p.  m.  inorganic  bodies. 

The  sputum  is  a  mixture  of  the  mucous  secretion  of  the  respiratory 
passages,  of  saliva  and  buccal  mucus.  Because  of  this  its  composition  is 
very  variable,  especially  under  pathological  conditions  when  various  pro- 
ducts mix  with  it.  The  chemical  constituents  are,  besides  the  mineral 
substances,  chiefly  mucin  with  a  little  proteid  and  nuclein  substance. 
Under  pathological  conditions  proteoses  and  peptone  (?),  which  are  prob- 
ably produced  by  bacterial  action  or  by  autolysis  (Wanner,  Simon  ^), 
volatile  fatty  acids,  glycogen,  Charcot's  crystals,  and  also  crystals  of 
cholesterin,  hsematoidin,  tyrosine,  fat  and  fatty  acids,  triple  phosphates, 
etc.,  have  been  found. 

The  form  constituents  are,  under  physiological  circumstances,  e  ■>ithe- 
lium-cells  of  various  kinds,  leucocytes,  sometimes  also  red  blood-corpuscles 
and  various  kinds  of  fungi.  In  pathological  conditions  elastic  fibres, 
spiral  formations  consisting  of  a  mucin-like  substance,  fibrin  coagulum, 
pus,  pathogenic  microbes  of  various  kinds,  and  the  above-mentioned 
crystals  occur. 

1  Pouchet,  Compt.  rend.,  96;  Freund,  cited  from  Maly's  Jahresber,  16,  471. 
^Schmidt,  cited  from  v.  Gorup-Besanez,  Lehrbuch,  4.  Aufl.,  727;  Oidtmann,  ifeid,, 
732. 

^Wanner,  Deutsch.  Arch.  f.  klin.  Med..  75;  Simon,  Arch.  f.  exp.  Path.  u.  Pharm.,  49. 


CHAPTER  XVIII. 

METABOLISM   WITH  VARIOUS   FOODS,   AND   THEIR   NECESSITY 

TO   MAX. 

The  conversion  of  chemical  energy  into  heat  and  mechanical  work 
which  characterizes  animal  life,  leads,  as  previously  stated  in  Chapter  I, 
to  the  formation  of  relatively  simple  compounds  —  carbon  dioxide,  urea, 
etc.  —  which  leave  the  organism,  and  which,  moreover,  being  very  poor  in 
energy,  are  for  this  reason  of  little  or  no  value  for  the  body.  It  is  there- 
fore absolutely  necessary  for  the  continuance  of  life  and  the  normal  course 
of  the  functions  of  the  body  that  the  organism  and  its  different  tissues 
should  be  supplied  with  new  material  to  replace  that  which  has  been 
exhausted.  This  is  accomplished  by  means  of  food.  Those  bodies  are 
designated  as  food  which  have  no  injurious  action  upon  the  organism  and 
which  serve  as  a  source  of  energy  and  can  replace  those  constituents  of 
the  body  that  have  been  consumed  in  metabolism  or  that  can  prevent  or 
diminish  the  consumption  of  such  constituents. 

Among  the  numerous  dissimilar  substances  which  man  and  animals 
take  with  the  food  all  cannot  be  equally  necessary  or  have  the  same  value. 
Some  perhaps  are  unnecessary,  while  others  may  be  indispensable.  We 
have  learned  Ijy  direct  observation  and  a  wide  experience  that  besides  the 
oxygen,  which  is  necessary  for  oxidation,  the  essential  foods  for  animals  in 
general,  and  for  man  especially,  are  water,  mineral  bodies,  proteins,  carbo- 
hydrates, and  fats. 

It  is  also  apparent  that  the  variovis  groups  of  foodstuffs  necessary  for 
the  tissues  and  organs  must  be  of  varying  importance;  thus,  for  instance, 
water  and  the  mineral  bodies  have  another  value  than  the  organic  foods, 
and  these  again  must  differ  in  importance  among  themselves.  The  knov.l- 
edge  of  the  action  of  various  nutritive  bodies  on  the  exchange  of  material 
from  a  qualitative  as  well  as  a  quantitative  point  of  view  must  be  of 
fundamental  importance  in  determining  the  value  of  different  nutritive 
substances  relative  to  the  demands  of  the  body  for  food  under  various  con- 
ditions, and  also  in  deciding  many  other  questions  —  for  instance,  the 
proper  nutrition  for  an  individual  in  health  and  in  disease. 

Such  knowledge  can  only  be  attained  by  a  series  of  systematic  and 
thorough  observations,  in  which  the  quantity  of  nutritive  material,  relative 

715 


716  METABOLISM. 

to  the  weight  of  the  body,  taken  and  absorbed  in  a  given  time  is  compared 
with  the  quantity  of  final  metabolic  products  which  leave  the  organism  at 
the  same  time.  Researches  of  this  kind  have  been  made  by  several  investi- 
gators, but  above  all  should  be  mentioned  those  made  by  Bischoff  and 
VoiT,  by  Pettenkofer  and  Voit,  and  by  Voit  and  his  pupils,  by  Rubner 
and  by  Atwater. 

It  is  absolutely  necessary  in  researches  on  the  exchange  of  material  to 
be  able  to  collect,  analyze,  and  quantitatively  estimate  the  excreta  of  the 
organism,  so  that  they  may  be  compared  with  the  quantity  and  composition 
of  the  nutritive  bodies  ingested.  In  the  first  place,  one  must  know  what 
the  habitual  excreta  of  the  body  are  and  in  what  way  these  bodies  leave  the 
organism.  One  must  also  have  trustworthy  methods  for  the  quantitative 
estimation  of  the  same. 

The  organism  may,  under  physiological  conditions,  be  exposed  to 
accidental  or  periodic  losses  of  valuable  material  —  such  losses  as 
only  occur  in  certain  individuals,  or  in  the  same  individual  only  at  a 
certain  period;  for  instance,  the  secretion  of  milk,  the  production  of 
eggs,  the  ejection  of  semen  or  menstrual  blood.  It  is  therefore  apparent 
that  these  losses  can  be  the  subject  of  investigation  and  estimation  only 
in  special  cases. 

The  regular  and  constant  excreta  of  the  organism  are  of  the  very 
greatest  importance  in  the  study  of  metabolism.  To  these  belong,  in  the 
first  place,  the  true  final  metabolic  products  —  carbon  dioxide,  urea  (uric 
acid,  hippuric  acid,  creatinine,  and  other  urinary  constituents),  and  a  part 
of  the  water.  The  remainder  of  the  water,  the  mineral  bodies,  and  those 
secretions  or  tissue  constituents  —  mucus,  digestive  fluids,  sebum,  perspira- 
tion, and  epidermal  formations  —  which  are  either  poured  into  the  intestinal 
tract,  or  secreted  from  the  surface  of  the  body,  or  broken  off  and  thereby 
lost  to  the  body,  also  belong  to  the  constant  excreta. 

The  remains  of  food,  sometimes  indigestible,  sometimes  digestible  but  not  acted 
upon,  which  are  contained  in  the  fseces,  and  which  vary  considerably  in  quantity 
and  composition  with  the  nature  of  the  food,  also  belong  to  the  excreta  of  the 
organism.  Even  though  these  remains,  which  are  never  absorbed  and  therefore  are 
never  constituents  of  the  animal  fluids  or  tissues,  cannot  be  considered  as  excreta 
of  the  body  in  a  strict  sense,  still  their  quantitative  estimation  is  absolutely  neces- 
sary in  certain  experiments  on  the  exchange  of  material. 

The  determination  of  the  constant  loss  is  in  some  cases  accompanied  with  the 
greatest  difficulties.  The  loss  from  the  detached  epidermis,  from  the  secretion  of 
the  .sebaceous  glands,  etc.,  cannot  be  determined  with  exactness  without  difficulty, 
and  therefore  —  as  they  do  not  occasion  any  appreciable  loss  because  of  their  small 
quantity  —  they  need  not  be  considered  in  quantitative  experiments  on  metabolism. 
This  also  applies  to  the  constituents  of  the  mucus,  bile,  pancreatic  and  intestinal 
juices,  etc.,  occurring  in  the  contents  of  the  intestine,  and  which,  leaving  the  body 
with  the  faeces,  cannot  be  sei)arated  from  the  other  contents  of  the  intestine  and 
therefore  cannot  be  quantitatively  determined  separately.  The  uncertainty  which 
because  of  the  intimated  difficulties,  attaches  itself  to  the  results  of  the  experiment. 


EXCRETA  OF  THE  ORGANISM.  717 

is  very  small  as  compared  to  the  variation  which  is  caused  by  different  individu- 
alities, different  modes  of  living,  different  foods,  etc.  Xo  general  but  only  apj^roxi- 
mate  values  can  therefore  be  given  for  the  constant  excreta  of  the  human  body. 

The  following  figures  represent  the  quantity  of  excreta  for  twenty-four 
hours  from  a  grown  man,  weighing  60-70  kilos,  on  a  mixed  diet.  The 
numbers  are  compiled  from  the  results  of  different  investigators. 

Grams. 

Water 2.500-3500 

Salts  (with  the  urine) 20-30 

Carbon  dioxide 750-900 

Urea 20-40 

Other  nitrogenous  urinary  constituents 2-5 

Solids  in  the  excrements 20-50 

These  total  excreta  are  approximately  divided  among  the  various 
excretions  in  the  following  way;  but  still  it  must  not  be  forgotten  tliat 
this  division  may  vary  to  a  great  extent  under  various  external  circum- 
stances: by  respiration  about  32  per  cent,  by  the  evaporation  from  the 
skin  17  per  cent,  with  the  urine  46-47  per  cent,  and  with  the  excrements 
5-9  per  cent.  The  eUmination  by  the  skin  and  lungs,  which  is  sometimes 
differentiated  by  the  name  "  perspiratio  insensibilis^'  from  the  visible 
elimination  by  the  kidneys  and  intestine,  is  on  an  average  about  50  per 
cent  of  the  total  elimination.  This  proportion,  quoted  only  relatively, 
is  subject  to  considerable  variation,  because  of  the  great  difference  in 
the  loss  of  water  through  the  skin  and  kidneys  under  different  circum- 
stances. 

The  nitrogenous  constituents  of  the  excretions  consist  chiefly  of  urea, 
or  uric  acid  in  certain  animals,  and  the  other  nitrogenous  urinary  con- 
stituents. A  disproportionately  large  part  of  the  nitrogen  leaves  the  body 
with  the  urine,  and,  as  the  nitrogenous  constituents  of  this  excretion  are 
final  products  of  the  metabolism  of  proteins  in  the  organism,  the  quantity 
of  proteins  catabolized  in  the  body  may  be  easily  calculated  by  multiplying 
the  quantity  of  nitrogen  in  the  urine  by  the  coefficient  6.25  Oj^  -=6.2b), 
if  it  is  admitted  that  the  proteins  contain  in  round  numbers  16  per  cent 
of  nitrogen. 

Still  another  question  is  whether  the  nitrogen  leaves  the  body  only  with 
the  urine  or  by  other  channels.  The  latter  is  habitually  the  case.  The  dis- 
charges from  the  intestine  always  contain  some  nitrogen,  which  as  stated 
in  Chapter  IX  consists  in  part  of  non-absorbed  remnants  of  the  food,  but 
in  chief  part  and  sometimes  entirely  of  constituents  of  the  epithelium  and 
the  secretions.  Under  these  circumstances  it  is  apparent  that  one  cannot 
give  any  exact  figures  which  are  valid  for  all  cases  for  that  part  of  the 
nitrogen  of  the  excrements  which  originates  from  the  digestive  tract  and 
from  the  digestive  fluids.  It  may  not  only  vary  in  different  individuals, 
but  also  in  the  same  individual  after  more  or  less  active  secretion  and 


718  METABOLISM. 

absorption.  In  the  attempts  made  to  determine  this  part  of  the  nitrogen 
of  the  excrements  it  has  been  found  that  in  man,  on  non-nitrogenous  or 
nearly  nitrogen-free  food,  it  amounts  in  round  numbers  to  somewhat  less 
than  1  gram  per  twenty-four  hours  (Rieder,  Rubner).  Even  with  such 
food  the  absolute  quantity  of  nitrogen  eliminated  by  the  faeces  increases 
with  the  quantity  of  food  because  of  the  accelerated  digestion  (Tsuboi  ',) 
and  is  greater  than  in  starvation.  Muller  ^  found  in  his  observations  on 
the  faster  Cetti  that  only  0.2  gram  nitrogen  was  derived  from  the  intes- 
tinal canal. 

The  quantity  of  nitrogen  which  leaves  the  body  under  normal  circum- 
stances by  means  of  the  hair  and  nails,  with  the  scaling  off  of  the  skin,  and 
with  the  perspiration  cannot  be  accurately  determined.  It  is  neverthe- 
less so  small  that  it  may  be  ignored.  Only  in  profuse  sweating  need  the 
elimination  by  this  channel  be  taken  into  consideration. 

The  view  was  formerly  held  that  in  man  and  carnivora  an  elimination 
of  gaseous  nitrogen  took  place  through  the  skin  and  lungs,  and  because  of 
this,  on  comparing  the  nitrogen  of  the  food  with  that  of  the  urine  and 
faeces,  a  nitrogen  deficit  occurred  in  the  visible  elimination. 

This  question  has  been  the  subject  of  much  discussion  and  of  numerous 
investigations.'  These  investigations  have  shown  that  the  above  assump- 
tion is  unfounded,  and  moreover  several  investigators,  especially  Petten- 
kofer  and  Voit,  and  Gruber,*  have  shown  by  experiments  on  man  and 
animals  that  with  the  proper  quantity  and  quality  of  food  the  body  can 
be  brought  into  nitrogenous  equilibrium,  in  which  the  quantity  of  nitrogen 
voided  with  the  urine  and  faeces  is  equal  or  nearly  equal  to  the  quantity 
contained  in  the  food.  Undoubtedly  we  must  admit  with  Voit  that  a 
deficit  of  nitrogen  does  not  exist,  or  it  is  so  insignificant  that  in 
experiments  upon  metabolism  it  need  not  be  considered.  Ordinarily, 
in  investigations  on  the  catabolism  of  proteins  in  the  body,  it  is 
only  necessary  to  consider  the  nitrogen  of  the  urine  and  faeces,  but 
it  must  be  remarked  that  the  nitrogen  of  the  urine  is  a  measure 
of  the  extent  of  the  catabolism  of  the  proteins  in  the  body,  while  the 
nitrogen  of  the  faeces  (after  deducting  about  1  gram  on  a  mixed  diet)  is 
a  measure  of  the  non-absorbed  part  of  the  nitrogen  of  the  food.  The 
nitrogen  of  the  food,  as  well  as  of  the  excreta,  is  generally  determined 
by  Kjeldahl's  method. 

^  Rieder,  Zeitschr.  f.  Biologie,  20;  Rubner,  ibvL,  15;  Tsuboi,  ibid.,  35. 

2  Berlin,  klin.  Wochenschr.,  1887. 

^See  Regnault  and  Reiset,  Annal.  d.  chim.  et  phys.  (3),  26,  and  Annal.  d.  Chem. 
u.  Pharm.,  73:  Seegen  and  Nowak,  Wien.  Sitzungsber.,  71,  and  Pfliiger's  Arch.,  25; 
Pettenkofer  and  Voit,  Zeitschr.  f.  Biologie,  16;  Leo,  Pfliiger's  Arch.,  26. 

*  Pettenkofer  and  Voit,  in  Hermann's  Handbuch,  6,  Thl.  1 ;  Griiber,  Zeitschr.  f. 
Biologie,  16  and  19. 


EXCRETION   OF  SULPHUR  AND   PHOSPHORUS.  719 

In  the  oxidation  of  the  proteins  in  the  organism  their  sulphur  is  oxidized 
into  sulphuric  acid,  and  on  this  depends  the  fact  that  the  elimination  of 
sulphuric  acid  hj  the  urine,  which  in  man  is  only  to  a  small  extent  derived 
from  the  sulphates  of  the  food,  makes  nearly  equal  variations  with  the 
elimination  of  nitrogen  by  the  urine.  If  the  amount  of  nitrogen  and  sul- 
phur in  the  proteins  is  considered  as  16  per  cent  and  1  per  cent  respectively, 
then  the  proportion  between  the  nitrogen  of  the  proteins  and  the  sulphuric 
acid,  H2SO4,  produced  by  their  combustion  is  in  the  ratio  5.2:1,  or  about 
the  same  as  in  the  urine  (see  page  622) .  The  determination  of  the  quantity 
of  sulphuric  acid  eliminated  in  the  urine  gives  us  an  important  means  of 
controlling  the  extent  of  the  transformation  of  proteins,  and  such  a  control 
is  especially  important  in  cases  in  which  it  is  expected  to  study  the  action 
of  certain  nitrogenous  non-albuminous  bodies  on  the  metabolism  of  pro- 
teins. A  determination  of  the  nitrogen  alone  is  not  sufficient  in  such 
cases.  A  perfectly  positive  measure  of  the  protein  catabolism  cannot  be 
made  from  the  sulphuric  acid  of  the  urine,  as  the  various  protein  sub- 
stances have  a  rather  variable  sulphur  content,  and  on  the  other  hand 
also  a  variable  quantity  of  the  sulphur  in  the  urine  exists  as  so-called 
neutral  sulphur. 

In  metabolism  experiments  the  total  sulphur  of  the  urine  as  well  as 
the  faeces  must  be  determined.  The  sulphur  of  the  catabolized  proteins 
is  quicker  eliminated,  according  to  v.  Wexdt,  than  the  nitrogen,  and  this 
behavior  of  sulphur  gives  a  more  positive  picture  of  the  temporal  cata- 
bolism of  protein  than  the  nitrogen.  This  is  all  the  more  important  as 
according  to  Falta  ^  not  only  does  the  nitrogen  corresponding  to  a  certain 
amount  of  protein  require  several  days  for  elimination  but  also  the  chief 
quantity  of  this  nitrogen  in  man  after  taking  different  kinds  of  proteins 
is  eliminated  with  varying  rapidity. 

Besides  lecithins  and  other  phosphatides  the  body  takes  with  its  food 
pseudonucleins  as  well  as  true  nucleins  and  these  are  absorbed  more  or 
less  completely  from  the  intestinal  tract  and  then  assimilated  (Gumlich, 
Saxdmeyer,  ^Iarcuse,  Rohmaxx,  and  Steixitz,  Loewi,-  and  others). 
On  the  other  hand,  the  phosphorized  protein  substances,  lecithins  and 
phosphatides,  are  also  decomposed  within  the  body,  and  their  phosphorus 
is  chiefly  eliminated  as  phosphoric  acid  and  also  in  part^  as  organic  phos- 

'  V.  Wendt,  Skand.  Arch.  f.  Physiol.,  17;  Falta,  Deutsch.  Arch.  f.  kliii.  Med.  86. 

^  In  regard  to  the  investigations  on  the  metabolism  of  phosphorus  and  the  methods 
used  therein,  see  Steinitz,  Pfliiger's  Arch.,  72;  Zadik,  ibid.,  77;  Leipziger,  ibid.,  78; 
Oertel  Zeitschr.  f.  physiol.  Chem.,  26;  Mandel  and  Oertel,  Bull.  Med.  Sciences,  N.  Y. 
Univ.,  1,  and  Ehrlich,  Inaug.-Diss..  Breslau,  1900:  Loewi,  Arch.  f.  exp.  Path.  u.  Pharm., 
45.  On  the  absorption  of  ca.sein,  see  Poda.  Prausnitz,  Micko,  and  P.  ^liiller,  2^itschr.  f. 
Biologic,  39.  The  literature  on  the  phosphorus  metabolism  can  be  found  in  Albu  and 
Neuberg,  Physiol,  u.  Pathol,  des  Mineralstoffwechsels,  Berlin,  1906. 


720  METABOLISM. 

phonis  (see  page  619).       For  these  reasons  the  phosphorus  is  of  great 
importance  in  certain  investigations  on  metabolism. 

If  it  is  found,  on  comparing  the  nitrogen  of  the  food  with  that  of  the 
urine  and  faeces,  that  there  is  an  excess  of  the  first,  this  means  that  the 
body  has  increased  its  stock  of  nitrogenous  substances  —  proteins.  If,  on 
the  contrary,  the  urine  and  fsBces  contain  more  nitrogen  than  the  food 
taken  at  the  same  time,  this  denotes  that  the  body  is  giving  up  part  of  its 
nitrogen  —  that  is,  a  part  of  its  own  proteins  has  been  decomposed.  We 
can,  from  the  quantity  of  nitrogen,  as  above  stated,  calculate  the  corres- 
ponding quantity  of  proteins  by  multiplying  by  6.25.^  Usually,  according 
to  Voit's  proposition,  the  nitrogen  of  the  urine  is  not  calculated  as  decom- 
posed proteins,  but  as  decomposed  muscle-substance  or  flesh.  Lean  meat 
contains  on  an  average  about  3.4  per  cent  nitrogen;  hence  each  gram  of 
nitrogen  of  the  urine  corresponds  in  round  numbers  to  about  30  grams  of 
flesh.  The  assumption  that  lean  meat  contains  3.4  per  cent  nitrogen  is 
arbitrary,  and  the  relationship  of  N:  C  in  the  proteins  of  dried  meat,  which 
is  of  great  importance  in  certain  experiments  on  metabolism,  is  given 
differently  by  various  experimenters,  namely,  1 :  3.22  —  1 : 3.68.  Argutin- 
SKY  found  in  beef,  after  complete  removal  of  fat  and  subtraction  of  glycogen, 
that  the  relationship  was  1:3.24  (see  Chapter  XI). 

The  carbon  leaves  the  body  chiefly  as  carbon  dioxide,  which  is  elimi- 
nated by  the  lungs  and  skin.  The  remainder  of  the  carbon  is  excreted  in 
the  urine  and  faeces  in  the  form  of  organic  compounds,  in  which  the  quan- 
tity of  carbon  can  be  determined  by  elementary  analysis.  It  used  to  be 
considered  sufficient  to  calculate  the  quantity  of  carbon  in  the  urine  from 
the  quantity  of  nitrogen  according  to  the  relationship  N:  C=  1:  0.67.  This, 
does  not  seem  to  be  trustworthy,  as  this  relationship  varies  and  depends, 
according  to  Tangl  and  PFLtJGER,  Lang.stein,  and  Steinitz,"  upon  the 
kind  of  food.  Tangl  has  shown  that  the  richer  the  food  is  in  carbohydrates 
the  more  carbon  and  heat  of  combustion  per  gram  of  nitrogen  does  the 
urine  contain.  He  found  the  following  for  1  gram  of  nitrogen  in  the 
urine:  With  diet  rich  in  fat  0.747  gram  C  and  9.22  Calories;  for  carbo- 
hydrate-rich diet  he  found  0.963  gram  C  and  11.67  Calories. 

The  quantity  of  gaseous  carbon  dioxide  eliminated  may  be  determined 
by  means  of  Pettenkofer's  respiration  apparatus  or  by  other  methods. 
By  multiplying  the  quantity  of  carbon  dioxide  found  by  0.273  one  obtains 
the  quantity  of  carbon  eliminated  as  CO^.  If  the  total  quantity  of  carbon 
eliminated  in  various  ways  is  compared  with  the  carbon  contained  in  the 

1  In  calculating  the  protein  catabolism  from  the  nitrogen  of  the  urine  it  must  not 
be  forgotten  that  the  food  often  contains  nitrogenous  extractions  whose  nitrogen  cannot 
be  calculated  as  protein  and  for  which  a  special  correction  must  be  made,  if  necessary. 

2  Tangl,  Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Supplbd.;  Pfluger  in  Pfiuger's  Arch.,  79; 
Langstein  and  Steinitz,  Centralbl.  f.  Physiol.,  19. 


CALCULATIOX   OF  THE   EXTENT   OF  CATABOLISM.  721 

food  some  idea  can  be  obtained  as  to  the  transformation  of  the  carbon 
compounds.  If  the  quantity  of  carbon  in  the  food  is  greater  than  in  the 
excreta,  then  the  excess  is  deposited;  while  if  the  reverse  be  the  case  it 
shows  a  corresponding  loss  of  body  substance. 

The  nature  of  the  substances  here  deposited  or  lost,  whether  they  con- 
sist of  proteins,  fats,  or  carbohydrates,  is  learned  from  the  total  quantity 
of  the  nitrogen  of  the  excretions.  The  corresponding  quantity  of  proteins 
may  be  calculated  from  the  quantity  of  nitrogen,  and,  as  the  average 
quantity  of  carbon  in  the  proteins  is  known,  the  quantity  of  carbon  which 
corresponds  to  the  decomposed  proteins  ma}-  be  easily  ascertained.  If 
the  quantity  of  carbon  thus  found  is  smaller  than  the  quantity  of  the  total 
carbon  in  the  excreta,  it  is  then  obvious  that  some  other  nitrogen-free  sub- 
stance has  been  consumed  besides  the  proteins.  If  the  quantity  of  carbon 
in  the  proteins  is  considered  in  round  numbers  as  53  per  cent,'  then  the 
relation  between  carbon  (53)  and  nitrogen  (16)  is  as  3.3  :  1.  If  the  total 
quantity  of  nitrogen  eliminated  is  multiplied  by  3.3,  the  excess  of  carbon 
in  the  eliminations  ovef  the  product  found  represents  the  carbon  of  the 
decomposed  non-nitrogenous  compounds.  For  instance,  in  the  case  of  a 
person  experimented  upon,  10  grams  of  nitrogen  and  200  grams  of  carbon 
were  eliminated  in  the  course  of  24  hours;  then  these  62.5  grams  of  protein 
correspond  to  33  grams  of  carbon,  and  the  difference.  200—  (3.3 X  10)  =  167, 
represents  the  quantity  of  carbon  in  the  decomposed  non-nitrogenous  com- 
pounds. If  we  start  from  the  simplest  case,  starvation,  where  the  body 
lives  at  the  expense  of  its  own  substance,  then,  since  the  quantity  of 
carbohydrates  as  compared  with  the  fats  of  the  body  is  extremely  small, 
in  such  cases  in  order  to  avoid  mistakes  the  assumption  must  be  made 
that  the  person  experimented  upon  has  used  only  fat  and  proteins.  As 
animal  fat  contains  on  an  average  76.5  per  cent  carbon,  the  quantity  of 

transformed  fat  may  be  calculated  by  multiplying  the  carbon  bv  :rT^  =  1-3. 

76.5 

In  the  case  of  the  above  example,  the  person  experimented  upon  would 

have  used  62.5  grams  of  proteins  and  167X1.3  =  217  grams  of  fat  of  his 

own  body  in  the  course  of  the  twenty-four  hours. 

Starting  from  the  nitrogen  balance,  it  can  be  calculated  in  the  same 
way  whether  an  excess  of  carbon  in  the  food  as  compared  with  the  quantity 
of  carbon  in  the  excreta  is  retained  by  the  body  as  proteins  or  fat  or  as 
both.  On  the  other  hand,  with  an  excess  of  carbon  in  the  excreta  one  can 
determine  how  much  of  the  loss  of  the  substance  of  the  body  is  due  to  a 
consumption  of  the  proteins  or  of  fat  or  of  both. 

The  quantity  of  water  and  mineral  bodies  voided  with  the  urine  and 
faeces  can  easily  be  determined.     The  quantity  of  water  eliminated  by  the 

*  This  figure  is  perhaps  a  little  too  high. 


722  METABOLISM. 

skin  and  lungs  may  be  directly  estimated  by  means  of  Pettenkofer 's 
apparatus.  The  quantity  of  oxygen  taken  up  is  calculated  as  the  difference 
between  the  weight  of  the  individual  before  the  experiment  plus  all  the 
directly  determined  substances  ingested,  and  the  final  weight  of  the  indi- 
vidual plus  all  his  excreta. 

The  oxygen  may  also  be  determined  directly,  according  to  RECiNAULT- 
Reiset's  method,  or  in  other  ways,  and  such  a  determination  with  the 
simultaneous  estimation  of  the  carbon  dioxide  eliminated  is  of  great 
importance  in  the  study  of  metabolism.^ 

On  comparing  the  inspired  and  the  expired  air  we  learn,  on  measuring 
them  when  dry  and  at  the  same  temperature  and  pressure,  that  the  volume 
of  the  expired  air  is  less  than  that  of  the  inspired  air.  This  depends  upon 
the  fact  that  not  all  of  the  oxygen  appears  again  in  the  expired  air  as  car- 
bon dioxide,  because  it  is  not  only  used  in  the  oxidation  of  carbon,  but 
also  in  part  in  the  formation  of  water,  sulphuric  acid,  and  other  bodies. 
The  volume  of  expired  carbon  dioxide  is  regularlv  less  than  the  volume  of 

CO 

the  inspired  oxygen,  and  the  relation  ——^,  which  is  called  the  respiratory 

quotient,  is  generally  less  than  1. 

The  magnitude  of  the  respiratory  quotient  is  dependent  upon  the  kind 
of  substances  destroyed  in  the  body.  In  the  combustion  of  pure  carbon 
one  volume  of  oxygen  yields  one  volume  of  carbon  dioxide,  and  the  quo- 
tient is  therefore  equal  to  1.  The  same  is  true  in  the  burning  of  carbo- 
hydrates, and  in  the  exclusive  decomposition  of  carbohydrates  in  the 
animal  body  the  respiratory  quotient  must  be  approximately  1.  In  the 
exclusive  metabolism  of  proteins  it  is  close  to  0.80,  and  with  the  decompo- 
sition of  fat  it  is  0.7.  In  starvation,  as  the  animal  draws  on  its  own  flesh 
and  fat,  the  respiratory  quotient  must  be  a  close  approach  to  the  latter 
figure.  The  respiratory  quotient  therefore  gives  important  data  on  the 
quality  of  the  material  decomposed  in  the  body,  naturally  Avith  the  suppo- 
sition that  the  elimination  of  carbon  dioxide,  independent  of  the  formation 
of  carbon  dioxide,  is  not  influenced  by  special  conditions,  such  as  the 
alteration  of  the  respiratory  mechanism. 

It  is  also  possible  in  systematized  experimentation  to  carry  on  the 
metabolism  experiments  so  that  the  decomposable  material  of  the  body, 
as  shown  by  the  respiratory  quotient,  remains  qualitatively  the  same,  at 

'  In  regard  to  the  methods  for  estimating  the  carbon-dioxide  excretion  and  the  oxy- 
gen consumption,  see  Zuntz,  Hermann's  Handbuch  d.  Physiol.,  4,  Tl.  2;  Hoppe-Seyler, 
Zeitschr.  f.  physiol.  Chem.,  19;  Sonden  and  Tigerstedt,  Skand.  Arch.  f.  Physiol.,  6; 
Speck,  Physiol,  des  menschl.  Atmens.  Leipzig,  1892;  Zuntz  and  Geppert,  Pfiuger's 
Arch.,  42:  Magnus-Levy,  ibid.,  55,  10,  where  the  works  of  Zuntz  and  his  pupils  are 
cited;  Hanriot  et  Richet,  Compt.  rend.,  104,  and  Atwater,  Bull,  of  Dept.  of  Agric, 
Washington,  Nos.  44,  63,  69,  and  109. 


CALORIFIC  VALUES  FROM   OXYGEN   CONSUMPTION.  723 

least  for  a  short  time.  In  such  experiments  it  has  been  shown,  especially 
by  ZuNTz  and  his  pupils/  that  the  extent  of  oxygen  consumption  may 
be  taken  as  a  measure  for  the  action  of  different  influences  on  the  extent  of 
metabolism.  This  possibility  is  based  on  the  fact  proved  by  Pfluger  and 
his  pupils,  and  by  Voit,-  that  the  consumption  of  oxygen  within  wide 
limits  is  independent  of  the  supply  of  oxygen,  and  is  exclusively  dependent 
upon  the  oxygen  demand  of  the  tissues.  For  certain  reasons  the  consump- 
tion of  oxygen  gives  indeed  a  better  conclusion  than  the  elimination  of 
carbon  dioxide  as  to  the  extent  of  exchange  of  material  and  energy;  but 
as  the  same  quantity  of  oxygen  (100  grams)  consumes  different  quantities 
of  fat,  carbohydrates,  and  proteins  in  the  body  —  namely,  35,  84.4,  and 
74.4  grams  respectively  —  the  respiratory  quotient  must  also  be  deter- 
mined, in  order  to  ascertain  the  nature  of  the  substance  burnt  in  the  body, 
simultaneously  with  the  determination  of  the  carbon  dioxide. 

As  the  different  foods  require  different  amounts  of  oxygen  in  the  com- 
bustion of  each  gram  of  substance  and  yield  different  amounts  of  CO,, 
each  gram  of  oxygen  taken  up  and  each  gram  of  carbon  in  the  expired 
!iir  as  carbon  dioxide  must  correspond  to  different  heat  values.  This 
follows  from  the  following  table: 

Calories  Calories 

per  grm.  C       Relative  pergrm.  Relative 

ill  the  CO2  of       Value.  Consumed  Value. 

the  Expired  Air.  Oxygen. 

In  the  combustion  of  cane-sugar  ..  .      9.5  100  3.56  118.6 

"     "  "  "meat 10.2  107  3.00  100.0 

"     "  "  "fat 12.3  129  3.27  109.0 

Pfluger  has  found  the  following  figures  for  the  calorific  value  of  1 
gram  oxygen: 

For  muscle  tissue  free  from  fat 3 .  30  Cal. 

Fat 3.29    " 

Starch •. 3.53     " 

The  figures  for  the  oxygen  differ,  as  seen  above,  le.ss  than  those  for  the  car- 
bon, and  this  is  the  reason  why,  as  above  stated,  the  oxygen  consumption  gives  a 
much  more  correct  conclusion  as  to  the  exchange  of  force  than  the  elimination  of 
carbon  dioxide.^ 

Kaufmann  *  encloses  the  individual  to  be  experimented  upon  in  a 
capacious  sheet-iron  room,  which  serves  both  as  a  respiration-chamber  and 
a  calorimeter,  and  which  permits  of  the  estimation  of  the  nitrogen  of  the 
urine  and  the]carbon  dioxide  expired,  as  well  as  the  inspired  oxygen  and  the 
quantity  of  heat  produced.     If  we  start  from  the  theoretically  calculated 

.'  See  footnote,  page  722. 

^Pfluger,  Pfiiiger's  Arch.,  6,  10,  and  14;  Finkler,  ibid.,  10;  Finkler  and  Oertmann, 
ibiii,  14;  Voit,  Zeitschr,  f.  Biologie,  11  and  14. 

'  See  Ad.  Magnus-Levj',  Pfiiiger's  Arch.,  55,  7,  and  Pfluger,  ihiil.,  77,  78,  and  79. 
*Arch.  d.  Physiologic  (5),  8. 


724  METABOLISM. 

formulae  for  the  various  possible  transformations  of  the  proteins,  fats,  and 
carbohydrates  in  the  body,  it  is  clear  that  other  values  must  be  obtained 
for  the  heat,  carbon  dioxide,  oxygen,  and  nitrogen  of  the  urine,  when  one, 
for  example,  admits  of  a  complete  combustion  of  proteins  to  urea,  carbon 
dioxide,  and  water,  or  of  a  partial  splitting  off  of  fat.  Another  relation- 
ship between  heat,  carbon  dioxide,  and  oxygen  is  also  to  be  expected  when 
the  fat  is  completely  burnt  or  when  it  is  decomposed  into  sugar,  carbon 
dioxide,  and  water.  In  this  way,  by  a  comparison  of  the  values  found  in 
special  cases  with  the  figures  calculated  for  the  various  transformations, 
Kaufmaxx  attempts  to  explain  the  various  decomposition  processes  in 
the  body  under  different  nutritive  conditions. 

I.     The  Energy  and  the  Relative  Nutritive  Value  of  Various 
Organic  Foodstuffs. 

With  the  organic  foods  the  organism  receives  a  suppl}'  of  chemical 
energy  which  is  converted  into  heat  and  mechanical  work  in  the  body. 
This  energy  of  the  various  foods  may  be  represented  by  the  amount  of  heat 
which  is  set  free  in  their  combustion.  This  quantity  of  heat  is  expressed  as 
calories,  and  a  small  calorie  is  the  quantity  of  heat  necessary  to  warm  1 
gram  of  water  from  0°  to  1°  C.  A  large  calorie  is  the  quantity  of  heat 
necessary  to  warm  1  kilo  of  water  1°  C.  Here  and  in  the  following  pages 
large  calories  are  to  be  understood.  There  are  numerous  investigations  by 
different  experimenters,  such  as  Fraxklaxd,  Daxilewski,  Rubxer, 
Berthelot,  Stohmaxx,  and  others,  on  the  calorific  value  of  different 
foodstuffs.  The  following  results,  which  represent  the  calorific  value  of  a 
few  nutritive  bodies  on  complete  combustion  outside  of  the  body  to  the 
highest  oxidation  products,  are  taken  from  Stohmaxx  's  ^  work. 

Calories. 

Casein 5 .  86 

Ovalbumin 5 .  74 

Conglutin 5 .  48 

Protein  (average) 5 .  71 

Animal  tissue-fat 9 .  50 

Butter-fat 9.23 

Cane-sugar 3 .  96 

Milk-sugar 3 .  95 

Dextrose 3 . 74 

Starch 4.19 

Fats  and  carbohydrates  are  completely  burnt  in  the  body,  and  one  can 
therefore  consider  their  combustion  equivalent  as  a  measure  of  the  living 

^  See  Rubner,  Zeitschr.  f.  Biologie,  21,  which  also  cites  the  works  of  Frankland  and 
Danilewski;  see  also  Berthelot,  Compt.  rend.,  102,  104,  and  110;  Stohmana.  Zeitschr. 
f.  Biologie,  31. 


CALORIFIC  VALUE   OF  THE   FOODSTUFFS.  725 

force  developed  by  them  within  the  organism.  We  generally  designate  9.3 
and  4.1  calories  for  each  gram  of  substance  as  the  average  for  the  physio- 
logical calorific  value  of  fats  and  carbohydrates  respectively. 

The  proteins  act  differently  from  the  fats  and  carbohydrates.  They 
are  only  incompletely  burnt,  and  they  yield  certain  decomposition  pro- 
ducts, which,  leaving  the  body  with  the  excreta,  still  represent  a  certain 
quantity  of  energy  which  is  lost  to  the  body.  The  heat  of  combustion  of 
the  proteins  is  smaller  within  the  organism  than  outside  of  it,  and  they 
must  therefore  be  specially  determined.  For  this  purpose  Rubxer  ^  fed  a 
dog  on  washed  meat,  and  he  subtracted  from  the  heat  of  combustion  of 
the  food  the  heat  of  combustion  of  the  urine  and  faeces,  which  corresponded 
to  the  food  taken  plus  the  quantity  of  heat  necessary  for  the  swelling  up  of 
the  proteins  and  the  solution  of  the  urea.  Rubxer  has  also  tried  to  deter- 
mine the  heat  of  combustion  of  the  proteins  (muscle-proteins)  decomposed 
in  the  body  of  rabbits  in  starvation.  According  to  these  investigations, 
the  physiological  heat  of  combustion  in  calories  for  each  gram  of  substance 
is  as  follows: 

1  grm.  of  the  dry  substance.  Calories. 

Protein  from  meat 4.4 

Muscle 4.0 

Protein  in  starvation .3.8 

Fat  (average  for  various  fats) 9.3 

Carbohydrates  (calculated  average) 4.1 

The  physiological  combustion  value  of  the  various  foods  belonging  to 
the  same  group  is  not  quite  the  same.  It  is,  for  instance,  3.97  calories  for 
a  vegetable  protein,  conglutin,  and  4.42  calories  for  an  animal  protein  body, 
syntonin.  According  to  Rubxer  the  normal  heat  value  per  1  gram  of 
animal  protein  may  be  considered  as  4.23  calories,  and  of  vegetable  protein 
as  3.96  calories.  When  a  person  on  a  mixed  diet  takes  about  60  per  cent 
of  the  proteins  from  animal  foods  and  about  40  per  cent  from  vegetable 
foods,  the  value  of  1  gram  of  the  protein  of  the  food  is  equivalent  to  about 
4.1  calories.  The  physiological  value  of  each  of  the  three  chief  groups  of 
organic  foods,  by  their  decomposition  in  the  body,  is  in  round  numbers  as 
follows: 

Calories. 

1  gram  protein 4  1 

1      "      fat 9.3 

1      "      carbohydrate 4.1 

As  will  be  shown,  the  fats  and  carbohydrates  may  decrease  the  metab- 
olism of  proteins  in  the  body,  while,  on  the  other  hand,  the  quantity  of 
proteins  in  the  body  or  in  the  food  acts  on  the  metabolism  of  fat  in  the 
body.  In  physiological  combustion  the  various  foods  may  replace  one 
another  to  a  certain  extent,  and  it  is  therefore  important  to  know  the 

'  Zeitschr.  f.  Biologie,  21. 


726  METABOLISM. 

ratio  of  replacement.  The  investigations  made  by  Rubner  have  taught 
that  this,  if  it  relates  to  the  force  and  heat  production  in  the  animal  body, 
is  a  proportion  that  corresponds  with  the  figures  of  the  heat  value  of  the 
same.  This  is  apparent  from  the  following  table.  In  this  is  found  the 
weight  of  the  various  foods  equal  to  100  grams  of  fat,  a  part  determined 
from  experiments  on  animals  and  a  part  calculated  from  figures  of  the  heat 
values. 

100  grams  fat  are  equal  to  or  isodynamic  with 

From  Experiments        From  the  Difference, 

on  Animals.  Heat  Value.  per  cent, 

Syntonin 225  213  +5.6 

Muscle-flesh  (dried) 243  235  +4  3 

Starch 232  229  +1.3 

Cane-sugar 234  235  -0 

Dextrose 256  255  -0 

From  the  given  isodynamic  value  of  the  various  foods  it  follows  that 
these  substances  replace  one  another  in  the  body  almost  in  exact  ratio  to 
the  energy  contained  in  them.  Thus  in  round  numbers  227  grams  of  pro-, 
tein  and  carbohydrate  are  equal  to  or  isodynamic  with  100  grams  of  fat  in 
regard  to  source  of  energy,  because  each  yields  930  calories  on  combustion 
in  the  body. 

By  means  of  recent  very  important  calorimetric  investigations  Rubner^ 
has  shown  that  the  heat  produced  in  an  animal  in  several  series  of  experi- 
ments extending  over  forty-five  days  corresponded  to  within  0.47  per  cent 
of  the  physiological  heat  of  combustion  calculated  from  the  decomposed 
body  and  foods.  Atwater  and  his  collaborators  ^  have  made  some  very 
thorough  investigations  on  this  subject  on  men.  In  their  experiments 
they  made  use  of  a  large  respiration  calorimeter,  which  not  only  deter- 
mined exactly  the  excreta  but  also  made  a  calorimetric  determination  of 
the  heat  given  out  by  the  person  experimented  upon,  i.e.,  the  work  per- 
formed. From  the  results  of  these  experiments  they  found  nearly  an 
absolutely  complete  agreement  between  the  calories  found  directly  and 
those  calculated. 

This  isodynamic  law  is  of  fundamental  value  in  the  study  of  metabo- 
lism and  nutrition.  By  this  law  it  is  possible  to  consider  the  processes  of 
metabolism  as  more  uniform  transformations  of  energy.  The  quantity 
of  energy  in  the  transformed  foods  or  the  constituents  of  the  body  may 
be  used  as  a  measure  for  the  total  consumption  of  energy,  and  the 
knowledge  of  the  quantity  of  energy  in  the  foods  must  also  be  the  basis 
for  the  calculation  of  dietaries  for  human  beings  under  various  conditions. 

1  Zeitschr.  f.  Biologic,  30. 

^  Bull,  of  Dept.  of  Agric,  Washington,  44,  63,  69,  and  109  and  Ergebnisse  des 
Physiologic  3. 


Total  loss 

Availability 

in  per  cent. 

in  per  cent. 

10.20 

89.8 

9.60 

90.4 

10.70 

89.3 

7.60 

92.4 

17.90 

82.1 

26.50 

73.5 

23.20 

76.8 

HEAT  VALUES  OF  THE  FOOD.  727 

The  heat  value  of  a  foodstuff  can  be  directly  determined  in  a  calori- 
meter but  may  also  be  calculated  from  its  composition.  If  one  subtracts 
from  the  gross  heat  value  of  the  food  obtained  in  one  way  or  another,  the 
combustion  heat  of  the  fseces  and  urine  with  the  same  diet,  there  is  obtained 
the  net  calorific  value  of  the  diet.  This  value,  calculated  in  percentage  of 
the  total  energy  content  of  the  food,  is  called  the  'physiological  availahility 
by  RuBNER.*  In  order  to  elucidate  this  we  will  give  a  few  of  Rubner's 
values.  The  loss  in  calories,  as  well  as  the  physiological  availability,  are 
calculated  in  percentages  of  the  total  energy  content  of  the  food. 

Loss  in  per  cent. 
Food.  In  urine.         In  the  freces. 

Cow's  milk 5.13  5.07 

Mixed  diet  (rich  in  fat) 3 .  87  5 .  73 

"         "     (poor  in  fat) 4.70  6.00 

Potatoes 2.00  5.60 

Graham  bread 2.40  15.50 

Rye  bread 2.20  24.30 

Meat 16.30  6.90 

In  order  to  simplify  the  calculation  of  the  energy  exchange  there  exist, 
besides  the  above-mentioned  standard  figures  for  the  physiological  calorific 
value  of  the  organic  foodstuffs,  also  for  the  carbon  of  the  carbon  dioxide, 
and  for  the  oxygen  other  standard  factors.  Thus  for  1  gram  of  meat 
(dry  substance)  free  from  fat  and  extractives  we  have  the  calculated 
value  of  5.44-5.77  Cal.  Kohler  -  found  5.678  Cal.  for  1  gram  of  ash 
and  fat-free  dried-meat  substance  of  the  ox  and  5.599  Cal.  for  the  horse. 
According  to  Frentzel  and  Schreuer  ^  45.4  Cal.  is  calculated  for  1  gram 
of  nitrogen  in  fat  and  ash-free  dried-meat  fseces  (dog),  while  6.97  to  7.45 
Cal.  is  calculated    for  1  gram  of  nitrogen  in    meat-urine.     The  calorific 

Cal 
urine  quotient  ■         seems  still,  as   above  given,  not   to  be  constant   for 

human  beings  at  least,  but  is  dependent  upon  the  variety  of  food. 

Instead  of  the  direct  determination  the  heat  of  combustion  can  also  be  deter- 
mined from  the  elementary  composition  according  to  the  following  principle  as 
suggested  by  E.  Voit.*  If  we  designate  the  heat  of  combustion  for  1  gram  of  the 
substance  by  Cal.  and  the  quantity  of  oxygen  necessary  for  the  complete  com- 
bustion of  1  gram  of  the  substance  ( =  oxygen  capacity  of  the  substance)  by  O, 

then  =K,  which  is  the  combustion  value  for  1  gram  of  oxygen.     The  oxygen 

capacity  can  be  calculated  from  the  elementary  composition,  and  when  the  value 
of  K  is  known,  the  combustion  heat  of  a  chemical  compound  or  a  known  mixture 

'  Zeitschr.  f.  Biologie,  42. 
^  Zeitschr.  f.  physiol.  Chem.,  31. 

^  The  works  of  Frentzel  and  Schreuer  may  be  found  in  Arch.  f.  (Anat.  u.)  Physiol., 
1901,  1902,  and  1903. 

*  Zeitschr.  f.  Biologie,  44.     See  also  Krummacher,  ibid. 


728  METABOLISM. 

can  be  readily  determined.  The  value  K  is  nearly  constant  for  substances  of  the 
same  groups;  but  also  different  groups  show  among  themselves  only  slight  devia- 
tion for  this  value.     Voit  obtained  the  following  values  for  a  few  of  the  foodstuffs: 

K.  (in  kg.  Cal.)  O  Capacity. 

Plant  protein 3 .  298  1 .740 

Animal  protein 3 .  273  '       1 .741 

Fat 3.271  2.863 

Carbohydrate 3 .  525  1 .  156 

These  methods  of  calculation  are,  according  to  Voit  and  Krummacher, 
admissible  for  practical  purposes. 

II.     Metabolism  in  Starvation. 

In  starvation  the  decomposition  in  the  body  continues  uninterruptedly^ 
though  with  decreased  intensity;  but,  as  it  takes  place  at  the  expense  of 
the  substance  of  the  body,  it  can  only  continue  for  a  limited  time.  When 
an  animal  has  lost  a  certain  fraction  of  the  mass  of  the  body  death  is  the 
result.  This  fraction  varies  with  the  condition  of  the  body  at  the  begin- 
ning of  the  starvation  period.  Fat  animals  succumb  when  the  weight  of 
the  body  has  sunk  to  one  half  of  the  original  weight.  Otherwise,  accord- 
ing to  Chossat,^  animals  die  as  a  rule  when  the  weight  of  the  body  has 
sunk  to  tw^o  fifths  of  the  original  weight.  The  period  when  death  occurs 
from  starvation  not  only  varies  with  the  varied  nutritive  condition  at  the 
beginning  of  starvation,  but  also  with  the  more  or  less  active  exchange  of 
material.  This  is  more  active  in  small  and  young  animals  than  in  large 
and  older  ones,  but  different  classes  of  animals  show  an  unequal  activity. 
Children  succumb  in  starvation  in  3-5  days  after  having  lost  one  fourth 
of  their  body  mass.  Grown  persons  may,  as  observed  upon  Succi,^  and 
other  professional  fasters,  starve  for  twenty  days  or  more  without  lasting 
injury;  and  there  are  reports  of  cases  of  starvation  extending  over  a 
period  of  even  more  than  forty  to  fifty  days.  Dogs  can  live  without  food 
from  four  to  eight  weeks,  birds  five  to  twenty  days,  snakes  more  than  half 
a  year,  and  frogs  more  than  a  year. 

In  starvation  the  weight  of  the  body  decreases.  The  loss  of  weight  is 
greatest  in  the  first  few  days,  and  then  decreases  rather  uniformly.  In 
small  animals  the  absolute  loss  of  weight  per  day  is  naturally  less  than 
in  larger  animals.  The  relative  loss  of  weight  —  that  is,  the  loss  of  weight 
of  the  unit  of  the  weight  of  the  body,  namely,  1  kilo  —  is,  on  the  contrary, 
greater  in  small  animals  than  in  larger  ones.  The  reason  for  this  is  that 
the  smaller  animals  have  a  greater  surface  of  body  in  proportion  to  their 
mass  than  larger  animals,  and  the  greater  loss  of  heat  caused  thereby  must 
be  replaced  by  a  more  active  consumption  of  material. 

*  Cited  from  Voit  in  Hermann's  Hnndbuch,  6,  Thl.  1,  100. 
^  See  Luciani,  Das  Hungern.     Hamburg  u.  Leipzig,  1890. 


STARVATION.  729 

It  follows  from  the  decrease  in  the  weight  of  the  body  that  the  absolute 
extent  of  metabolism  must  diminish  in  starvation.  If,  on  the  contrary, 
the  extent  of  the  metabolism  is  referred  to  the  unit  of  the  weight  of  the 
body,  namely,  1  kilo,  it  appears  that  this  quantity  remains  nearly 
unchanged  during  starvation.  The  investigations  of  Zuntz,  Lehmann, 
and  others^  on  the  professional  faster  Cetti  showed  on  the  third 
and  sixth  days  of  starvation  an  average  consumption  of  4.65  c.c. 
oxygen  per  kilo  in  one  minute,  and  on  the  ninth  to  eleventh  day  an 
average  of  4.73  c.c.  The  calories,  as  a  measure  of  the  metabohsm,  fell 
on  the  first  to  fifth  day  of  starvation  from  1850  to  1600  calories,  or 
from  32.4  to  30  per  kilo,  and  it  remained  nearly  unchanged,  if  referred  to 
the  unit  of  body  weight.^ 

The  extent  of  the  metabolism  of  proteins,  or  the  elimination  of  nitrogen 
by  the  urine,  which  is  a  measure  of  the  same,  diminishes  as  the  weight  of 
the  body  diminishes.  This  decrease  is  not  regular  or  the  same  during 
the  entire  period  of  starvation,  and  the  extent  depends,  as  the  experiments 
made  upon  carnivora  have  shown,  upon  several  circumstances.  During 
the  first  few  days  of  starvation  the  excretion  of  nitrogen  is  greatest,  and 
the  richer  the  body  is  in  protein,  due  to  the  food  previously  taken,  the 
greater  is  the  protein  catabolism  or  the  nitrogen  elimination,  according 
to  VoiT.  The  nitrogen  elimination  diminishes  the  more  rapidly  —  that  is, 
the  curve  of  the  decrease  is  more  sudden  —  the  richer  in  proteins  the  food 
was  which  was  taken  before  starvation.  This  condition  is  apparent  from 
the  following  table  of  data  of  three  different  starvation  experiments  made 
by  VoiT  ^  on  the  same  dog.  This  dog  received  2500  grams  of  meat  daily 
before  the  first  series  of  experiments,  1500  grams  of  meat  daily  before 
the  second  series,  and  a  mixed  diet  relatively  poor  in  nitrogen  before  the 
third  series. 

Day  of  Starvation.  Grams  of  Urea  Eliminated  in  Twenty-four  Hours. 

Ser.  I.  Ser.  II.  Ser  III 

First 60.1  26.5  13.8 

Second 24.9  18.6  11.5 

Third 19.1  15.7  10.2 

Fourth 17.3  14.9  12.2 

Fifth 12.3  14.8  12.1 

Sixth 13.3  12.8  12.6 

Seventh 12.5  12.9  11.3 

Eighth 10.1  12.1  10.7 

In  man  and  also  in  animals  sometimes  a  rise  in  the  nitrogen  excretion 
is  observed  about  the  second  or  third  starvation  day,  which  is  then  fol- 
lowed by  a  regular  diminution.     This  rise  is  explained  by  Prausnitz, 

1  Berlin,  klin.  Wochenschr.,  1887. 

^  See  also  Tigerstedt  and  collaborators  in  Skand.  Arch.  f.  Physiol.,  7. 

'See  Hermann's  Handbuch,  6,  Thl.  1,  89. 


730  METABOLISM. 

TiGERSTEDT,  Landergren,^  as  follows:  At  the  commencement  of  star- 
vation the  protein  metaboUsm  is  reduced  by  the  glycogen  still  present 
in  the  body.  After  the  consumption  of  the  glycogen,  which  takes  place  in 
great  part  during  the  first  days  of  starvation,  the  destruction  of  proteins 
increases  as  the  glycogen  action  decreases,  and  then  decreases  again  when 
the  body  has  become  poorer  in  available  proteins. 

Other  conditions,  such  as  varying  quantities  of  fat  in  the  body,  have 
an  influence  on  the  rapidity  with  which  the  nitrogen  is  eliminated  during 
the  first  days  of  starvation.  After  the  first  few  days  of  starvation  the 
ehmination  of  nitrogen  is  more  uniform.  It  may  diminish  gradually  and 
regularly  until  the  death  of  the  animals  or  there  may  be  a  rise  in  the  last 
days,  a  so-called  premortal  increase.  Whether  the  one  or  the  other 
occurs,  depends  upon  the  relationship  between  the  protein  and  fat  content 
of  the  body. 

Like  the  destruction  of  proteins  during  starvation,  the  decomposition 
of  fat  proceeds  uninterruptedly,  and  the  greatest  part  of  the  calories  needed 
during  starvation  are  supplied  by  the  fats.  According  to  Rubner  and 
VoiT  the  protein  catabolism  varies  only  slightly  in  starving  animals  at 
rest  and  at  an  average  temperature,  and  forms  a  constant  portion  of  the 
total  exchange  of  energy;  of  the  total  calories  in  dogs  10-16  per  cent  comes 
from  the  protein  decomposition  and  84-90  per  cent  from  the  fats.  This  is 
at  least  true  for  starving  animals  which  had  a  sufficiently  great  original 
fat  content.  If  on  account  of  starvation  the  animal  has  become  relatively 
poorer  in  fat  and  the  fat  content  of  the  body  has  fallen  below  a  certain 
Hmit,  then  in  order  to  supply  the  calories  necessary  a  larger  quantity  of 
protein  is  destroyed  and  the  premortal  increase  now  occurs  (E.   Voit  ^) , 

Since  the  fat  has  a  diminishing  influence  on  the  destruction  of  proteins 
corresponding  to  what  was  said  above,  the  elimination  of  nitrogen  in  star- 
vation is  less  in  fat  than  in  lean  individuals.  For  instance,  only  9  grams 
of  urea  were  voided  in  twenty-four  hours  during  the  later  stages  of  starva- 
tion by  a  well-nourished  and  fat  person  suffering  from  disease  of  the  brain, 
while  I.  MuNK  found  that  20-29  grams  urea  were  voided  daily  by  Cetti,^ 
who  had  been  poorly  nourished. 

The  investigations  on  the  exchange  of  gas  in  starvation  have  shown,  as 
previously  mentioned,  that  the  absolute  extent  of  the  same  is  diminished, 
but  that  when  the  consumption  of  oxygen  and  elimination  of  carbon 
dioxide  are  calculated  on  the  unit  of  weight  of  the  body,  1  kilo,  this  quantity 

'  Prausnitz,  Zeitschr.  f.  Biologie,  29;  Tigerstedt  and  collaborators,  1.  c;  Landergren, 
" Undersokningar  ofver  menniskans  agghviteomsattning,  Inaug.-Diss.  Stockholm, 
1902. 

^Zeitschr.  f.  Biologie,  41,  167  and  502.  See  also  Kaufmann,  ibid.,  and  N.  Schulz, 
ibid.,  and  Pfliiger's  Arch.,  76. 

3  Berl.  klin.  Wochenschr.,  1887. 


STARVATION.  731 

quickly  sinks  to  a  minimum  and  then  remains  unchanged,  or,  on  the  con- 
tinuation of  the  starvation,  may  actually  rise.  It  is  a  well  known  fact 
that  the  body  temperature  of  starving  animals  remains  nearly  constant, 
without  showing  any  appreciable  decrease,  during  the  greater  part  of  the 
starvation  period.  The  temperature  of  the  animal  first  sinks  a  few  days 
before  death,  and  death  occurs  at  about  33-30°  C. 

From  what  has  been  said  about  the  respiratory  quotient  it  follows  that 
in  starvation  it  is  about  the  same  as  with  fat  and  meat  exclusively  as  food, 
i.e.,  approximately  0.7.  This  is  often  the  case,  but  it  may  occasionally  be 
lower,  0.65-0.50,  as  observed  in  the  cases  of  Cetti  and  Succi.  As  explana- 
tion for  this  unexpected  behavior  one  must  admit  of  a  storage  of  incom- 
pletely oxidized  substances  in  the  body  during  starvation. 

Water  passes  uninterruptedly  from  the  body  in  starvation  even  when 
none  is  taken.  If  the  quantity  of  water  in  the  tissues  rich  in  proteins  is 
considered  as  70-80  per  cent,  and  the  quantity  of  proteins  in  the  same 
20  per  cent,  then  for  each  gram  of  protein  destroyed  about  4  grams  of 
water  are  set  free.  This  liberation  of  water  from  the  tissues  is  generally 
sufficient  to  supply  the  loss  of  water,  and  starvation  is  ordinarily  not 
accompanied  with  thirst.  Starving  animals,  as  a  rule,  do  not  partake  of 
water. 

The  loss  of  water  calculated  on  the  percentage  of  the  total  organism  must 
natuially  be  essentially  dependent  upon  the  previous  amount  of  fatty  tissue  in  the 
body.  If  we  bear  these  conditions  in  mind,  then,  it  seems,  according  to  Boht- 
LiNGK,!  that,  from  experiments  upon  white  mice,  the  animal  body  is  poorer  in 
water  during  inanition.  The  body  loses  more  water  than  is  set  free  by  the  destruc- 
tion of  the  tissues. 

The  mineral  substances  leave  the  body  uninterruptedly  in  starvation 
until  death,  and  the  influence  of  the  destruction  of  tissues  is  plainly  per- 
ceptible by  their  elimination.  Because  of  the  destruction  of  tissues  rich  in 
potassium  the  proportion  between  potassium  and  sodium  in  the  urine 
changes  in  starvation,  so  that,  contrary  to  the  normal  conditions,  the 
potassium  is  eliminated  in  proportionately  greater  quantities.  INIunk  also 
observed  in  Cetti  's  ^  case  a  relative  increase  in  the  phosphoric  acid  and 
calcium  in  the  urine  during  starvation,  which  was  due  to  an  increased 
exchange  of  bone-substance. 

Contrary  to  the  above  Bohtlingk  with  starving  white  mice,  and  Katsuyama^ 
with  starving  rabbits  found  a  greater  excretion  of  sodium  than  potassium. 

The  question  as  to  the  participation  of  the  different  organs  in  the  loss 
of  weight  of  the  body  during  starvation  is  of  special  interest.     In  elucida- 

1  Arch,  des  sciences  biol.  de  St.  Petersbourg,  5. 

2  L,  c. 

^  Bohtlingk,  1.  c;  Katsuyama,  Zeitschr.  f.  physiol.  Cheni.,  26. 


732  METABOLISM. 

tion  of  this  point  we  give  the  following  results  of  Chossat's  experiments 
on  pigeons,  and  those  of  Voit  ^  on  a  male  cat.  The  results  are  percentages 
of  weight  lost  from  the  original  weight  of  the  organ. 

Pigeon  (Chossat).  Male  Cat  (Voir). 

Adipose  tissue 93  per  cent.  97  per  cent. 

Spleen 71  "  "  67  "  " 

Pancreas 64  "  "  17  "  " 

Liver 52  "  "  54  "  " 

Heart   45  "  "  3  "  " 

Intestine 42  "  "  18  "  " 

Muscles 42  "  "  31  "  " 

Testicles —  "  "  40  "  " 

Skin 33  "  "  21  "  " 

Kidneys 32  "  "  26  "  " 

Lungs 22  "  '  18  "  " 

Bones  17  "  "  14  "  " 

Ner\'OUs  system 2  "  "  3  "  " 

The  total  quantity  of  blood,  as  well  as  the  quantity  of  solids  contained 
therein,  decreases,  as  Panum  and  others  ^  have  shown,  in  the  same  propor- 
tion as  the  weight  of  the  body.  The  statements  in  regard  to  the  loss 
of  water  by  different  organs  are  somewhat  contradictory;  according  to 
LuKJANOW  ^  it  seems  that  the  various  organs  act  somewhat  differently  in 
this  respect. 

The  above-tabulated  results  cannot  serve  as  a  measure  of  the  metabo- 
lism in  the  various  organs  during  starvation.  For  instance,  the  nervous 
system  shows  only  a  small  loss  of  weight  as  compared  with  the  other 
organs,  but  from  this  it  must  not  be  concluded  that  the  exchange  of 
material  in  this  system  of  organs  is  least  active.  The  condition  may  be 
quite  different;  for  one  organ  may  derive  its  nutriment  during  starvation 
from  some  other  organ  and  exist  at  its  expense.  A  positive  conclusion 
cannot  be  drawn  in  regard  to  the  activity  of  the  metabolism  in  an  organ 
from  the  loss  of  weight  of  that  organ  in  starvation.  Death  by  starvation 
is  not  the  result  of  the  death  of  all  the  organs  of  the  body,  but  it  depends 
more  likely  upon  the  disturbance  in  the  nutrition  of  a  few  less  vitally 
important  organs  (E.  Voit^). 

In  calculating  or  determining  the  loss  of  weight  of  the  organs  in  star- 
vation the  original  fat  content  of  the  organs  must  also  be  considered. 
With  the  consideration  of  the  fat  content  of  the  organs,  determined  or 
estimated  in  a  special  way  before  the  starvation  period  and  at  the  end, 
E.  Voit  ^  has  found  the  following  loss  of  weight  in  the  supposed  fat-free 

'  Cited  from  Voit  in  Hermann's  Handbuch,  6,  Part  1,  96  and  97. 

^  Panum,  Virchow's  Arch.,  29:  London,  Arch.  d.  scienc.  biol.  de  St.  P^tersbourg,  4. 

^  Zeitschr.  f.  physiol.  Chem.,  13. 

*  Zeitschr.  f.  Biologie,  41. 

^Ibid.,  46. 


STAR\'ATIOX.  733 

organs  in  starvation,  namely,  muscles  41  per  cent,  viscera  42  per  cent, 
skin  28  per  cent,  and  skeleton  5  per  cent. 

The  knowledge  of  metabolism  during  starvation  is  of  the  greatest 
importance  in  the  study  of  nutrition,  and  it  forms  to  a  certain  extent  the 
starting-point  for  the  study  of  metabolism  under  different  physiological 
and  pathological  conditions.  To  answer  the  question  whether  the 
metabolism  of  a  person  in  a  special  case  is  abnormally  increased 
or  diminished  it  is  naturally  xery  important  to  know  the  average 
extent  of  metabolism  of  a  healthy  person  under  the  same  circumstances, 
for  comparison.  This  quantity  can  be  called  the  starvation  requirement, 
that  is,  the  extent  of  metabolism  used  in  absolute  bodily  rest 
and  inactivity  of  the  intestinal  tract.  As  a  measure  of  this  quantity 
we  determine,  according  to  Geppert-Zuxtz,  the  extent  of  gaseoiLs 
exchange,  and  especially  the  consumption  of  oxygen,  of  a  person 
lying  down,  best  sleeping,  in  the  early  morning  and  at  least  twelve 
hours  after  a  light  meal  not  rich  in  carbohydrates.  The  gas  volume 
reduced  to  0°  C.  and  760  mm.  Hg  pressure  is  calculated  on  1  kilo  of  body 
weight  and  for  one  minute.  The  results  varv'  between  3  and  4. .5  cc.  for 
the  consumption  of  oxygen,  and  between  2.5  and  3.5  cc.  for  the  carbon 
dioxide.  As  average  3.81  cc.  oxygen  and  3.08  cc  carbon  dioxide  are 
usually  given.  ^ 

The  extent  of  protein  destruction  cannot  be  detennined  in  transient 
experiments,  and  for  the.se  reasons  only  the  values  found  after  several 
days  of  starvation  are  useful.  In  the  star\^ation  experiments  on  Cetti 
and  .Succi  the  elimination  of  nitrogen  per  kilo  on  the  fifth  to  the  tenth  star- 
vation day  was  0.150-0.202  gram  X.  In  a  recent  starvation  experiment 
made  by  E.  and  O.  Freuxd  -  upon  Succi  the  nitrogen  excretion  on  the 
twenty-first  day  sank  to  2.82  grams  X.  The  portion  of  the  urea  nitrogen 
of  the  total  nitrogen  sank  from  85-89  per  cent  on  the  first  days 
of  starvation  to  73  per  cent  on  the  fifteenth  day  and  56-54  per 
cent  on  the  day  before  the  last  day  of  starvation.  Xone  of  the 
other  nitrogenous  constituents  examined  increased  to  the  samo 
extent  as  the  urea  decreased.  The  amount  of  neutral  sulphur  rose 
from  10  to  40  per  cent  of  the  total  sulphur.  In  a  recent  series 
of  inve.stigations  upon  the  faster  Succi.  Brugsch^  found  on  the 
twenty-first  to  the  thirtieth  day  that  the  urea  only  amounted  to  54-69.4 
per  cent  of  the  total  nitrogen  while  the  quantity  of  ammonia,  because 
of  a  high  acidosis,  rose  to  15.4-35.3  per  cent.  The  amino-acid  fraction 
was  also  above  normal. 

'  See  V.  Xoorden.  Lehrbuch  der  Pathlogie  des  Stoffwechsel,  Berlin,  1906. 
^Wien.  klin.  Rundschau.  1901.  Xos.  .5  and  6. 
^  Zeitschr.  f.  exp.  Path.  u.  Therap.  1. 


734  METABOLISM. 

III.    Metabolism  with  Inadequate  Nutrition. 

The  food  may  be  quantitatively  insufficient,  and  the  final  result  is 
absolute  inanition.  The  food  may  also  be  qualitatively  insufficient  or,  as 
we  say,  inadequate.  This  occurs  when  any  of  the  necessary  nutritive 
bodies  are  absent  in  the  food,  while  the  others  occur  in  sufficient  or  perhaps 
even  in  excessive  amounts. 

Lack  of  Water  in  the  Food.  The  quantity  of  water  in  the  organism  is 
greatest  during  foetal  life,  and  then  decreases  with  increasing  age.  Natu- 
rally, the  quantity  differs  in  various  organs.  The  tissue  in  the  body  being 
poorest  in  water  is  the  enamel,  which  is  almost  free,  containing  only  2  p.  m. 
water,  the  teeth  about  100  p.  m.,  the  fatty  tissues  60-120  p.  m.  The 
bones,  with  140-440  p.  m.,  and  the  cartilage,  with  540-740  p.  m.,  are 
somewhat  richer  in  water,  while  the  muscles,  blood,  and  glands,  with  750 
to  more  than  800  p.  m.,  are  still  richer.  The  quantity  of  water  is  even 
greater  in  the  animal  fluids  (see  preceding  chapter),  and  the  adult  body 
contains  in  all  about  630  p.  m.  water.*  If  it  is  borne  in  mind  that  two 
thirds  of  the  animal  organism  consists  of  water;  that  water  is  of  the  very 
greatest  importance  in  the  normal,  physical  composition  of  the  tissues; 
moreover  that  all  flow  of  juices,  all  exchange  of  substance,  all  supply  of 
nutrition,  all  increase  or  destruction,  and  all  discharge  of  the  products  of 
destruction,  are  dependent  upon  the  presence  of  water;  and,  in  addition, 
that  by  its  evaporation  it  is  an  important  regulator  of  the  temperature  of 
the  body,  we  perceive  that  water  must  be  necessary  for  life.  If  the  loss 
of  water  be  not  replaced  by  fresh  supplies  sooner  or  later,  the  organism 
succumbs  and  death  may  occur  earlier  with  lack  of  water  than  with  com- 
plete inanition  (Landauer,  Nothwang). 

If  the  water  is  withdrawn  for  a  certain  time,  as  Landauer  and  espe- 
cially W.  Straub  have  shown,  it  has  an  accelerating  influence  upon  the 
decomposition  of  protein.  This  increased  destruction  has,  according  to 
Landauer,  the  purpose  of  replacing  a  part  of  the  water  withheld  (by  means 
of  the  increased  metabolism).  The  deprivation  of  water  for  a  short  time 
may,  according  to  Spiegler,^  especially  in  man,  cause  a  diminution  in 
the  protein  metabolism  by  means  of  a  reduced  protein  absorption. 

Lack  of  Mineral  Substances  in  the  Food.  In  a  previous  chapter  atten- 
tion was  called  in  several  instances  to  the  importance  of  the  mineral  bodies 
and  also  to  the  occurrence  of  certain  mineral  substances  in  certain  amounts 
in  the  various  organs.  The  mineral  content  of  the  tissues  and  fluids  is 
not  very  great  as  a  rule.     With  the  exception  of  the  skeleton,  which  con- 

'  See  Voit  in  Hennann's  Handbuch,  6,  Tl.  I,  345. 

^Landauer,    Maly's    Jahresber.,   24;    Nothwang,  Arch.    f.    Hygiene,    1892;    Straub, 
Zeitschr.  f.  Biologic,  37  and  38;  Spiegler,  ibiil.,  40. 


LACK   OF  MINERAL   SUBSTANCES   IN   THE   FOOD.  735 

tains  about  220  p.  m.  mineral  bodies  (Volkmann '),  the  animal  fluids  or 
tissues  are  poor  in  inorganic  constituents,  and  the  quantity  of  these 
amounts,  as  a  rule,  only  to  about  10  p.  m.  Of  the  total  quantity  of  min- 
eral substances  in  the  organism,  the  greatest  part  occurs  in  the  skeleton, 
830  p.  m.,  and  the  next  greatest  in  the  muscles,  about  100  p.  m.  (Volk- 
mann)  . 

The  mineral  bodies  seem  to  be  partly  dissolved  in  the  fluids  and  partly 
combined  with  organic  substances.  In  accordance  with  this  the  organism 
persistently  retains,  with  food  poor  in  salts,  a  part  of  the  mineral  sub- 
stances, also  such  as  are  soluble,  as  the  chlorides.  On  the  burning  of  the 
organic  substances  the  mineral  bodies  combined  therewith  are  set  free  and 
may  be  eliminated.  It  is  also  admitted  that  they  in  part  combine  with 
the  new  products  of  the  combustion,  and  in  part  with  organic  nutritive 
bodies  poor  in  salts  or  nearly  salt-free,  which  are  absorbed  from  the  intes- 
tinal canal  and  are  thus  retained  (Voit,  Forster^). 

If  this  statement  is  correct,  it  is  possible  that  a  constant  supply  of 
mineral  substances  with  the  food  is  not  absolutely  necessary,  and  that  the 
amount  of  inorganic  bodies  which  must  be  administered  is  insignificant. 
The  question  whether  this  be  so  or  not  has  not,  especially  in  man,  been 
sufficiently  investigated;  but  generally  we  consider  the  need  of  mineral 
substances  by  man  as  very  small.  It  may,  however,  be  assumed  that 
man  usually  takes  with  his  food  a  considerable  excess  of  mineral  substances. 

Experiments  to  determine  the  action  of  an  insufficient  supply  of  min- 
eral substances  with  the  food  in  animals  have  been  made  by  several  inves- 
tigators, especially  Forster.  He  observed,  on  experimenting  with  dogs 
and  pigeons  with  food  as  poor  as  possible  in  mineral  substances,  that  a 
very  suggestive  disturbance  of  the  functions  of  the  organs,  particularly 
the  muscles  and  the  nervous  system,  appeared,  and  that  death  resulted  in 
a  short  time,  earlier  in  fact  than  in  complete  starvation.  On  observations 
made  upon  himself  Taylor  ^  found  on  partaking  less  than  0. 1  gram 
salts  per  diem  that  the  chief  disturbance  occurred  in  the  muscular  system. 

BuNGE  in  opposition  to  these  observations  of  Forster's  has  suggested 
that  the  early  death  in  these  cases  was  not  caused  by  the  lack  of  mineral 
salts,  but  more  likely  by  the  lack  of  bases  necessary  to  neutralize  the 
sulphuric  acid  formed  in  the  combustion  of  the  proteins  in  the  organism; 
these  bases  must  then  be  taken  from  the  tissues.      In  accordance  with 


^  See  Voit  in  Hermann's  Handbuch,  6,  Part  1,  353. 

^  Forster,  Zeitschr.  f.  Biologie,  9.  See  also  Voit  in  Hermann's  Handbuch,  6,  Part  1, 
354.  In  regard  to  the  occurrence  and  the  behavior  of  the  various  mineral  constituents 
of  the  animal  body  see  the  work  of  Albu  and  Neuberg,  Physiologic  and  Pathologie  des 
Mineralstoffwechsel,  Berlin,  190G. 

^  University  of  Cahfornia  Publications,  Pathol.  1. 


736  METABOLISM. 

this  view,.  Bunge  and  Lunin  ^  also  found,  in  experimenting  with  mice, 
that  animals  which  received  nearly  ash-free  food  with  the  addition  of 
sodium  carbonate  were  kept  alive  twice  as  long  as  those  which  had  the 
same  food  without  the  sodium  carbonate.  Special  experiments  also  show 
that  the  carbonate  cannot  be  replaced  by  an  equivalent  amount  of  sodium 
chloride,  and  that  to  all  appearances  it  acts  by  combining  with  the  acids 
formed  in  the  body.  The  addition  of  alkali  carbonate  to  the  otherwise 
nearly  ash-free  food  may  indeed  delay  death,  but  cannot  prevent  it,  and 
even  in  the  presence  of  the  necessary  amount  of  bases  death  results  for 
lack  of  mineral  substances  in  the  food. 

In  the  above  series  of  experiments  made  by  Bunge  the  food  of  the 
animal  consisted  of  casein,  milk-fat,  and  cane-sugar.  While  milk  alone 
was  an  adequate  and  sufficient  food  for  the  animal,  Bunge  found  that  the 
animal  could  not  be  kept  alive  longer  by  food  consisting  of  the  above  con- 
stituents of  milk  and  cane-sugar  with  the  addition  of  all  the  mineral  sub- 
stances of  milk  than  with  the  food  mentioned  in  the  above  experiments 
with  the  addition  of  alkali  carbonate.  The  question  whether  this  result 
is  to  be  explained  by  the  fact  that  the  mineral  bodies  of  milk  are  chem- 
ically combined  with  the  organic  constituents  of  the  same  and  can  be 
assimilated  only  in  such  combinations,  or  whether  it  depends  on  other 
conditions,  Bunge  leaves  undecided.  These  observations,  however,  show 
how  difficult  it  is  to  draw  positive  conclusions  from  experiments  made 
thus  far  with  food  poor  in  salts.  Further  investigations  on  this  subject 
seem  to  be  necessary. 

With  an  insufficient  supply  of  chlorides  with  the  food  the  elimination 
of  chlorine  by  the  urine  decreases  constantly,  and  at  last  it  may  stop 
entirely,  while  the  tissues  still  persistently  retain  the  chlorides.  It  has 
already  been  stated  (Chapter  IX)  how  chloride  starvation  influences  other 
functions,  especially  the  secretion  of  gastric  juice.  If  there  be  a  lack  of 
sodium  as  compared  with  potassium,  or  if  there  be  an  excess  of  potassium 
compounds  in  any  other  form  than  KCl,  the  potassium  combinations  are 
replaced  in  the  organism  by  NaCl,  so  that  new  potassium  and  sodium 
compounds  are  produced  which  are  voided  with  the  urine.  The  organism 
becomes  poorer  in  NaCl,  which  therefore  must  be  taken  in  greater  amounts 
from  the  outside  (Bunge).  This  occurs  habitually  in  herbivora,  and  in 
man  with  vegetable  food  rich  in  potash.  For  human  beings,  and  especially 
for  the  poorer  classes  of  people  who  live  chiefly  on  potatoes  and  foods 
rich  in  potash,  common  salt  is,  under  these  circumstances,  not  only  a 
condiment,  but  a  necessary  addition  to  the  food  (Bunge  2).  On  the  be- 
havior of  chlorides,  especially  sodium  chloride,  in  the  animal  body  as  well 

'  Bunge,  Lehrbuch  der  phybiol.  Chem.,  4.  Aufl.,  97;  Lunin,  Zeitschr.  f.  physiol. 
Chem.,  5. 

^  Zeitschr.  f.  Biologic  9. 


LACK  OF  MINERAL  SUBSTANCES  IN  THE   FOOD.  737 

as  the  elimination  or  the  retention  of  NaCl  in  diseases  we  have  an  abundance 
of  investigations,  which  may  be  found  in  Albu  and  Neuberg's  work 
previously  cited. 

Lack  of  Alkali  Carbonates  or  Bases  in  the  Food.  The  chemical  processes 
in  the  organism  are  dependent  upon  the  presence  of  tissue-fluids  of  a  cer- 
tain reaction,  and  this  action,  which  is  habitually  alkaline  towards  litmus 
and  neutral  towards  phenolphthalein,  is  chiefly  due  to  the  presence  of 
alkali  carbonates  and  carbon  dioxide.  The  alkali  carbonates  are  also  of 
great  importance  not  only  as  a  solvent  for  certain  protein  bodies  and  as 
constituents  of  certain  secretions,  such  as  the  pancreatic  and  intestinal 
juices,  but  they  are  also  a  means  of  transportation  of  the  carbon  dioxide 
in  the  blood.  It  is  therefore  easy  to  understand  that  a  decrease  below 
a  certain  point  in  the  quantity  of  alkali  carbonate  must  endanger  life. 
Such  a  decrease  not  only  occurs  with  lack  of  bases  in  the  food  which  brings 
about  various  disturbances  and  death  by  a  relatively  great  production  of 
acids  through  the  burning  of  the  proteins,  but  it  also  occurs  when  an  animal 
is  given  dilute  mineral  acids  for  a  period.  The  importance  of  ammonia  as 
a  means  of  neutralizing  the  acids  produced  or  introduced  into  the  body 
as  well  as  the  different  resistance  of  man  and  other  animals  towards  this 
action  of  acids  has  already  been  discussed  in  Chapter  XV. 

Lack  of  Phosphates  and  Earths.  With  the  exception  of  the  importance 
of  the  alkaline  earths  as  carbonates  and  more  especially  as  phosphates  in 
the  physical  composition  of  certain  structures,  such  as  the  bones  and  teeth, 
their  physiological  importance  is  nearly  unknown.  The  importance  of 
calcium  for  certain  enzymotic  processes  and  also  the  great  importance  of 
calcium  ions  for  the  functions  of  the  muscles  and  especially  for  cell  life 
give  an  indication  of  the  great  importance  of  the  alkaline  earths  for  the 
animal  organism.  Very  little  is  known  in  regard  to  the  need  of  these 
earths  in  adults,  and  no  average  results  can  be  given  for  this.  The  same 
is  true  for  the  need  of  phosphates  or  phosphoric  acid,  whose  great  impor- 
tance is  recognized  not  only  for  the  construction  of  the  bones  but  also 
for  the  functions  of  the  muscles,  the  nervous  system,  the  glands,  the  organs 
of  generation,  etc.  The  extent  of  this  need  is  most  difficult  to  determine 
as  the  body  shows  a  strong  tendency,  when  increased  amounts  of  phos- 
phorus are  introduced,  to  retain  more  than  is  necessary.  The  need  of 
phosphates  is  relatively  smaller  in  adults  than  in  young,  developing  ani- 
mals, and  in  these  latter  the  question  of  the  action  of  insufficient  supply 
of  earthy  phosphates  and  alkaline  earths  upon  the  bone  tissue  is  of  special 
interest.  In  regard  to  this  question  we  refer  to  Chapter  X  and  to  the 
cited  work  of  Albu-Neuberg. 

Another  important  question  is.  How  far  do  the  phosphates  take  part 
in  the  construction  of  the  phosphorized  constituents  of  the  body  or  to 


738  METABOLISM. 

what  extent  are  they  necessary?  The  experiments  of  Rohmann  and  his 
pupils  '  with  phosphorized  (casein,  vitelhn)  and  non-phosphorized  pro- 
teins (edestin)  and  phosphates  show  that  with  the  introduction  of  casein 
and  vitelhn  a  deposition  of  nitrogen  and  phosphorus  takes  place,  while 
with  non-phosphorized  protein  and  phosphates  this  does  not  seem  to  occur. 
The  body  apparently  does  not  have  the  power  of  building  up  the  phos- 
phorized cell  constituents  necessary  for  cell  life  from  non-phosphorized 
proteins  and  phosphates.  On  the  contrary,  according  to  the  observations 
of  several  investigators,  the  lecithins  seem  to  possess  this  power.  As 
known  from  the  investigations  of  Miescher,  the  development  of  genera- 
tive organs  of  the  salmon  which  are  very  rich  in  nuclein  substances  and 
phosphatides  from  the  muscles  which  are  relatively  poor  in  organic-com- 
bined phosphorus  seem  to  indicate  a  synthesis  of  phosphorized  organic 
substance  from  the  phosphates.  Other  investigators,  such  as  v.  Wendt,^ 
also  admit  of  a  synthesis  of  phosphorized  protein  substances  by  the  aid 
of  inorganic  phosphates. 

Lack  of  Iron.  As  iron  is  an  integral  constituent  of  haemoglobin,  abso- 
lutely necessary  for  the  introduction  of  oxygen,  just  so  is  it  an  indispen- 
sable constituent  of  food.  Iron  is  a  never-failing  constituent  of  the 
nucleins  and  nucleoproteids,  and  herein  lies  also  another  reason  for  the 
necessity  of  the  introduction  of  iron.  Iron  is  also  of  great  importance  for 
the  action  of  certain  enzymes,  the  oxidases.  In  iron  starvation  iron  is 
continually  eliminated,  even  though  in  diminished  amounts;  and  with  an 
insufficient  supply  of  iron  with  the  food  the  formation  of  haemoglobin 
decreases.  The  formation  of  haemoglobin  is  not  only  enhanced  by  the 
supply  of  organic  iron,  but  also,  according  to  the  general  view,  by  inor- 
ganic iron  preparations.  The  various  divergent  statements  on  this  ques- 
tion have  already  been  given  in  a  previous  chapter  (on  the  blood). 

In  the  absence  of  'protein  bodies  in  the  food  the  organism  must  nourish 
itself  by  its  own  protein  substances,  and  with  such  nutrition  it  must  sooner 
or  later  succumb.  By  the  exclusive  administration  of  fat  and  carbohy- 
drates the  consumption  of  proteins  in  these  cases  is  very  considerably 
reduced.  According  to  the  doctrine  of  C.  Voit,  which  has  been  defended 
by  recent  investigations  of  E.  Voit  and  Korkunoff,^  the  protein  metab- 
olism is  never  so  low  under  these  conditions  as  in  starvation.  Accord- 
ing to  several  investigators,  such  as  Hirschfeld,  Kumagawa,  Klem- 
PERER,  Siven,   I;Andergren,^  and  others,  the  protein  metabolism  may 

'  See  Marcuse,  Pfliiger's  Arch.,  67,  and  footnote  2,  page  719. 

^Skand.  Arch.,  f.  Physiol.  17. 

5  Zeitschr.  f.  Biologic,  32. 

*  Hirschfeld,  Virchow's  Arch.,  Ill;  Kumagawa,  ifnd.,  116;  Klemperer,  Zeitschr. 
f.  khn.  Med.,  16;  Sivon,  Skand.  Arch.  f.  Physiol.,  10 and  11;  Landergren,  1.  c.  11;  footnote 
1,  page  730.     See  also  iMaly's  Jahresber.,  32.    . 


ARTIFICIAL   MIXTURES.  739 

indeed,  with  exclusively  fat  and  carbohydrate  diet,  be  smaller  than  in 
complete  starvation.  Thus  Landergren  has  observed  on  an  adult, 
healthy  man  in  nitrogen  starvation  but  with  sufficient  supply  of  energy 
(about  40  calories  per  1  kilo  as  carbohydrates  and  fat)  on  the  fourth  star- 
vation day  that  the  nitrogen  excretion  was  not  more  than  4  grams.  On 
the  seventh  day,  with  only  carbohydrates,  the  nitrogen  excretion  sank  to 
3.34  grams,  which  corresponded  to  0.047  gram  N  per  kilo  of  body  weight 
and  to  0.29  gram  protein. 

The  absence  of  fats  and  carbohydrates  in  the  food  affect  carnivora  and 
herbivora  somewhat  differently.  It  is  not  known  whether  carnivora  can 
be  kept  alive  for  any  length  of  time  by  food  entirely  free  from  fat  and  car- 
bohydrates.^ But  it  has  been  positively  demonstrated  that  they  can  be 
kept  alive  a  long  time  by  feeding  exclusively  with  meat  freed  as  much  as 
possible  from  visible  fat  (Pflucjer^).  Human  beings  and  herbivora,  on 
the  contrary,  cannot  live  for  any  length  of  time  on  such  food.  On  one 
side  they  lose  the  property  of  digesting  and  assimilating  the  necessarily 
large  amounts  of  meat,  and  on  the  other  a  distaste  for  large  quantities  of 
meat  or  proteins  soon  appears. 

A  question  of  greater  importance  is  whether  it  is  possible  to  maintain 
life  in  an  animal  for  any  length  of  time  with  a  mixture  of  simple  organic 
and  inorganic  foodstuffs.  This  was  not  possible  in  the  experiments  of 
BuNGE  and  Lunin,  cited  above.  Later  investigators,  such  as  Hall  and 
Steinitz,  Falta  and  Noeggerath,  arrived  at  somewhat  better  results; 
and  RoHMANN  ^  has  arrived  at  still  more  conclusive  results.  He  used 
mice  in  his  experiments,  and  fed  them  with  a  mixture  of  casein,  white  of 
egg,  vitellin,  potato-starch,  wheat-starch,  margarine,  and  salts.  With 
this  diet  the  animals  maintained  their  body  weight  and  brought  forth 
young.  These  latter  could  not  be  raised  on  artificial  food.  A  better 
result  was  obtained  by  adding  some  malt  to  the  food.  It  was  also  possible 
to  further  raise  with  artificial  food  to  maturity'  mice  which  had  been 
formed  and  born  with  artificial  food.  These  mice  remained  somewhat 
smaller  than  the  normal,  and  no  living  young  could  be  obtained  from 
them.  If  we  exclude  the  fact  that  the  foodstuffs  fed  were  not  all  simple 
(white  of  egg,  malt),  pure  foods  it  seems  as  if  artificial  mixtures  of  food 
are  sufficient  to  maintain  at  least  an  adult  animal  for  a  long  time,  while 
it  is  not  quite  sufficient  for  the  development  of  a  young  animal. 

'  See  Horbaczewski,  Maly's  Jahresber.,  31,  715. 

'  Pfliiger's  Arch.,  50. 

^  Hall,  Arch.  f.  (Anat.  u.)  Physiol.,  1896;  Steinitz,  Uber  Versuche  mit  kiinstlicher 
Ernahrung,  Inaug.-Diss.,  Breslau,  1900;  Falta  and  Noeggerath,  Hofmeister;  Beitrage, 
7;  Rohmann,  Klin,  therap.  Wochenschr.,  1902,  No.  40. 


'40  METABOLISM. 


IV.  Metabolism  with  Various  Foods. 

For  the  carnivora,  as  above  stated,  meat  as  poor  as  possible  in  fat  may 
be  a  complete  and  sufficient  food.  As  the  proteins  moreover  take  a  special 
place  among  the  organic  nutritive  bodies  by  the  quantity  of  nitrogen  they 
contain,  it  is  proper  that  we  first  describe  the  metabolism  with  an  exclu- 
sivel}'  meat  diet. 

Metabolism  with  food  rich  in  proteins,  or  feeding  only  with  meat  as 
poor  in  fat  as  possible. 

By  an  increased  supply  of  proteins  their  catabolism  and  the  elimina- 
tion of  nitrogen  is  increased,  and  this  in  proportion  to  the  suppl}-  of  pro- 
teins. 

If  a  certain  quantity  of  meat  has  been  given  to  carnivora  as  food  daily 
and  the  quantity  is  suddenly  increased,  an  augmented  catabolism  of  pro- 
teins, or  an  increase  in  the  quantity  of  nitrogen  eliminated,  is  the  result. 
If  the  animal  is  fed  daily  for  a  certain  time  with  larger  quantities  of  the 
same  meat,  a  part  of  the  proteins  accumulates  in  the  body,  but  this  part 
decreases  from  day  to  day.  while  there  is  a  corresponding  daily  increase 
in  the  elimination  of  nitrogen.  In  this  way  a  nitrogenous  equilibrium  is 
established;  that  is,  the  total  quantity  of  nitrogen  eliminated  is  equal  to 
the  quantity  of  nitrogen  in  the  absorbed  proteins  or  meat.  If,  on  the 
contrary,  an  animal  which  is  in  nitrogenous  equilibrium,  having  been  fed 
on  large  quantities  of  meat,  is  suddenly  given  a  small  quantity  of  meat  per 
clay,  then  the  animal  uses  up  its  own  body  proteins,  the  amount  decreasing 
from  day  to  day.  The  elimination  of  nitrogen  and  the  catabolism  of 
proteins  decrease  constantly,  and  the  animal  may  in  this  case  also  pass 
into  nitrogenous  equilibrium,  or  nearly  into  this  condition.  These  rela- 
tions are  illustrated  by  the  following  table  {Yoit  ^) : 

Grams  of  Meat  in  the  Food  per  Day. 


1 

Before  the  Test. 
500 

During  the  Test. 
1500 

2 

1500 

1000 

Grams  of  Flesh  Metabolized  in  Body  per  Day. 

1 
1222 
1153 

1310             1390             1410             1440 
1086             1088             1080             1027 

6 
1450 

7 
1500 

In  the  first  case  (1)  the  metabolism  of  meat  before  the  beginning  of  the 
actual  experiment  on  feeding  with  500  grams  of  meat  was  447  grams,  and 
it  increased  considerably  on  the  first  day  of  the  experiment,  after  feeding 
with  1500  grams  of  meat.  In  the  second  case  (2),  in  which  the  animal  was 
previously  in  nitrogenous  equilibrium  with  1500  grams  of  meat,  the  meta- 


»  Hermann's  Handbuch,  6,  Part  I,  110. 


METABOLISM  WITH  FOOD  RICH  IX   PROTEINS.  741 

bolism  of  flesh  on  the  first  day  of  the  experiment,  with  only  1000  grams 
meat,  decreased  considerably,  and  on  the  fifth  day  nearly  a  nitrogenous 
equilibrium  was  obtained.  During  this  time  the  animal  gave  up  daily 
some  of  its  own  proteins.  Between  that  point  below  which  the  animal 
loses  from  its  own  weight  and  the  maximum,  which  seems  to  be  depend- 
ent upon  the  digestive  and  assimilative  capacity  of  the  intestinal  canal,  a 
carnivora  may  be  kept  in  nitrogenous  equilibrium  with  varying  quantities 
of  proteins  in  the  food. 

The  supply  of  proteins,  as  well  as  the  protein  condition  of  the  body^ 
affects  the  extent  of  the  protein  metabolism.  A  body  which  has  become 
rich  in  proteins  by  a  previous  abundant  meat  diet  must,  to  prevent  a  lo.ss 
of  proteins,  take  up  more  protein  with  the  food  than  a  body  poor  in  pro- 
teins. 

In  regard  to  the  rapidity  with  which  the  protein  catabolism  takes  place 
Falta  *  has  found  in  man  but  not,  or  at  least  not  to  the  same  extent,  in 
dogs,  that  quite  great  differences  exist  between  the  different  proteins. 
Thus  on  feeding  pure  proteins  the  chief  amount  of  the  nitrogen  is  much 
quicker  eliminated  after  feeding  casein  than  after  genuine  ovalbumin. 
This  latter  is  much  easier  demolished  after  a  previous  denaturization  by 
coagulation  than  in  the  native  state,  which  indicates  that  an  unequal  resist- 
ance of  the  different  proteins  towards  the  digestive  juices  plays  a  part. 
Even  on  feeding  with  easily  decomposable  proteins  it  takes  always  several 
days  before  the  total  nitrogen  corresponding  thereto  is  eliminated,  which 
depends  according  to  Falta  upon  a  progressive  demolition  of  the  protein. 
From  the  unequal  rapidity  with  which  the  different  proteins  are  decom- 
posed it  follows  that  in  the  passage  from  a  diet  poor  in  protein  to  one  rich 
in  protein  the  time  within  which  nitrogenous  equilibrium  occurs  depends 
chiefly  upon  the  kind  of  protein  contained  in  the  food. 

Pettenkofer  and  Voit  have  made  investigations  on  the  metabolism 
of  fat  with  an  exclusively  protein  diet.  These  investigations  have  shown 
that  by  increasing  the  quantity  of  proteins  in  the  food  the  daily  meta- 
bolism of  fat  decreases,  and  they  have  drawn  the  conclusion  from  these 
experiments,  as  detailed  in  Chapter  X,  that  even  a  formation  of  fat  may 
take  place  under  these  circumstances.  The  objections  presented  by 
PFLtJGER  to  these  experiments,  as  well  as  the  proofs  of  the  formation  of 
fat  from  proteins,  are  also  given  in  the  above-mentioned  chapter. 

According  to  Pfll-ger's  doctrine  the  protein  can  influence  the  forma- 
tion of  fat  only  in  an  indirect  way,  namely,  in  that  it  is  consumed  instead 
of  the  non-nitrogenous  bodies  and  hence  the  fat  and  fat-forming  carbo- 
hydrates are  spared.  If  sufficient  protein  is  introduced  into  the  food  to 
satisfy  the  total  nutritive  requirements,  then  the  decomposition  of  fat 

1  Deutsch.  Arch.  f.  klin.  Med.  86. 


742  METABOLISM. 

stops;  and  if  non-nitrogenous  food  is  taken  at  the  same  time,  this  is  not 
consumed,  but  is  stored  up  in  the  animal  body,  the  fats  as  such,  and  the 
carbohydrates  at  least  in  great  part  as  fat. 

Pfluger  defines  the  "  nutritive  requirement  "  as  the  smallest  quantity 
of  lean  meat  which  produces  nitrogenous  equilibrium  without  causing  any 
decomposition  of  fat  or  carbohydrates.  At  rest  and  at  an  average  tem- 
perature it  is  found  for  dogs  to  be  2.073  to  2.099  grams  of  nitrogen  ^  (in 
meat  fed)  per.kilo  of  flesh  weight  (not  body  weight,  as  the  fat,  which  often 
forms  a  considerable  fraction  of  the  weight  of  the  body,  cannot  as  it  were 
be  used  as  dead  measure).  Even  when  the  supply  of  protein  is  in  excess 
of  the  nutritive  requirements,  PFLiJGER  has  found  that  the  protein  meta- 
bolism increases  with  an  increased  supply  until  the  limit  of  digestive  power 
is  reached,  which  limit  is  about  2600  grams  of  meat  with  a  dog  weighing 
30  kilos.  In  these  experiments  of  Pflxjoer's  all  of  the  excess  of  protein 
introduced  was  not  completely  decomposed,  but  a  part  was  retained  by 
the  body.  Pfluger  therefore  defends  the  proposition  "that  a  supply  of 
proteins  only,  without  fat  or  carbohydrate,  does  not  exclude  a  protein 
fattening." 

From  what  has  been  said  on  protein  metabolism  in  starvation  and  with 
exclusive  protein  food  it  follows  that  the  protein  catabolism  in  the  animal 
body  never  stops,  that  the  extent  is  dependent  in  the  first  place  upon  the 
extent  of  protein  supph',  and  that  the  animal  body  has  the  property, 
within  wide  limits,  of  accommodating  the  protein  catabolism  to  the  pro- 
tein supply. 

These  and  certain  other  peculiarities  of  protein  catabolism  have  led 
YoiT  to  the  view  that  all  proteins  in  the  body  are  not  decomposed  with 
the  same  ease.  "S'oit  differentiates  the  protein  fixed  in  the  tissue-elements, 
so-called  organized  proteins,  tissue-protei^is,  from  those  proteins  which 
circulate  with  the  fluids  in  the  body  and  its  tissues  and  which  are  taken  up 
by  the  living  cells  of  the  tissues  from  the  interstitial  fluids  washing  them 
and  are  destro^'ed.  These  circulating  'proteins  are,  according  to  Voit, 
more  easily  and  quickly  destroyed  than  the  tissue-proteins.  When, 
therefore,  in  a  fasting  animal  which  has  been  previously  fed  with  meat  an 
abundant  and  quickly  decreasing  decomposition  of  proteins  takes  place, 
while  in  the  further  course  of  starvation  this  protein  catabolism  becomes 
less  and  more  uniform,  this  depends  upon  the  fact  that  the  supply  of 
circulating  proteins  is  destroyed  chiefly  in  the  first  days  of  starvation  and 
the  tissue-proteins  in  the  last  days. 

The  tissue-elements  constitute  an  apparatus  of  a  relatively  stable 
nature,  which  have  the  power  of  taking  proteins  from  the  fluids  washing 
the  tissues  and  appropriating  them,  while  their  own  proteins,  the  tissue- 

*  See  SchondorfF,  Pfliiger's  Arch.,   71. 


TISSUE   AND   CIRCULATIXG   PROTEINS.  743 

pr>;teins,  are  ordinarily  catabolized  to  only  a  small  extent,  about  1  per 
cent  daily  (Voit).  By  an  increased  supply  of  proteins  the  activity  of 
the  cells  and  their  ability  to  decompose  nutritive  proteins  is  also  increased 
to  a  certain  degree.  When  nitrogenous  equilibrium  is  obtained  after  an 
increased  supply  of  proteins,  it  denotes  that  the  decomposing  power  of 
the  cells  for  proteins  has  increased  so  that  the  same  quantity  of  proteins 
is  metabolized  as  is  supplied  to  the  body.  If  the  protein  metabolism  is 
decreased  by  the  simultaneous  administration  of  other  non-nitrogenous 
foods  (see  below),  a  part  of  the  circulating  proteids  may  have  time  to 
become  fixed  and  organized  by  the  tissues,  and  in  this  way  the  mass  of 
the  flesh  of  the  body  increases.  During  starvation  or  with  a  lack  of  pro- 
teins in  the  food  the  reverse  takes  place,  for  a  part  of  the  tissue  proteins 
is  converted  into  circulating  proteins  which  are  metabolized,  and  in  this 
case  the  flesh  of  the  body  decreases. 

Voit's  theory  has  been  criticised  by  several  investigators  and  espe- 
cially by  PFLtJGER.  Pfluger's  statement,  based  on  an  investigation  made 
by  one  of  his  pupils,  Schoxdorff,^  is  that  the  extent  of  protein  destruction 
is  not  dependent  upon  the  quantity  of  circulating  proteins,  but  upon  the 
nutritive  condition  of  the  cells  for  the  time  being  —  a  view  which  is  not 
verv  contradictory  of  ^'oit  if  the  author  does  not  misunderstand  Pflij- 
GER.  YoiT  ^  has,  as  is  known,  stated  that  the  conditions  for  the  destruc- 
tion of  substances  in  the  body  exist  in  the  cells,  and  also  that  the  circu- 
lating protein,  likewise  according  to  Voit,  is  first  catabolized  after  having 
been  taken  up  by  the  cells  from  the  fluids  washing  them.  The  point  of 
Yoit's  theory  is  that  all  proteins  are  not  destroyed  in  the  body  with  the 
same  degree  of  readiness.  The  organized  protein,  which  is  fixed  by  the 
cells  and  has  become  a  part  of  the  same,  is  destroyed  less  readily,  accord- 
ing to  Voit,  than  the  protein  taken  up  by  the  cells  from  the  nutritive  fluid, 
which  serves  as  material  for  the  chemical  construction  of  the  very  much 
more  complicated  organized  proteins.  This  nutritive  protein,  which  cir- 
culates with  the  fluids  before  it  is  taken  up  by  the  cells,  and  which  can 
exist  in  store  in  the  cells  as  well  as  in  the  fluids,  agreeably  to  Voit's  view, 
has  been  called  circulating  protein  or  supply  protein  by  him.  It  is  clear 
that  these  names  may  lead  to  misunderstanding,  and  therefore  too  much 
stress  should  not  be  put  upon  them.  The  most  essential  part  of  Voit's 
theory  is  the  supposition  that  the  food  protein  of  the  cells  is  more  easily 
destroyed  than  the  organized,  real  protoplasmic  protein,  and  this  asser- 
tion can  hardly,  for  the  present,  be  considered  as  refuted  or  exactly 
proved. 


1  Pfliiger,  Pfliiger's  Arch.,  54:  Schondorff,  ibid.,  51. 
^  Zeitschr.  f.  Biologic,  11. 


744  METABOLISM. 

The  investigations  in  recent  years,  especially  those  of  Folin,  which 
show  that  the  amount  of  certain  nitrogenous  urinary  constituents,  such  as 
creatinine,  uric  acid  and  the  combinations  containing  neutral  sulphur  are 
nearly  independent  upon  the  quantity  of  portein  taken  as  food,  while  the 
quantity  of  urea  is  determined  by  the  protein  partaken  of,  speaks  without 
any  doubt  in  favor  of  Voit 's  view  that  we  must  differentiate  between  the 
real  cell  protein  and  the  food  protein.  This  has  also  led  Folin  to  differen- 
tiate between  endogenous  and  exogenous  protein  metabolism.  The  ex- 
perience on  protein  feeding  and  the  endeavor  of  the  body,  as  observed 
by  ScHREUER,^  on  going  to  an  ordinary  diet  after  abundant  protein  feeding, 
to  remain  at  the  old  state  previous  to  the  over  feeding  of  protein,  speak 
also  for  the  fact  that  protein  retained  by  the  body  is  not  quite  the  same 
as  the  other  body  protein. 

This  question  is  intimately  connected  with  another,  namely,  whether 
the  food  proteins  taken  up  by  the  cells  are  metabolized  as  such  or  whether 
they  are  first  organized,  i.e.,  are  converted  into  specific  cell  protein.  The 
investigations  of  Panum  and  Falck  and  others  ^  on  the  transitory  prog- 
ress of  the  elimination  of  urea  after  a  meal  rich  in  proteins  throws  light 
on  this  question.  From  experiments  upon  a  dog  it  was  found  that  the 
elimination  of  urea  increases  almost  immediately  after  a  meal  rich  in  pro- 
teins, and  that  it  reaches  its  maximum  in  about  six  hours,  when  about  one 
half  of  the  quantity  of  nitrogen  corresponding  to  the  administered  proteins 
is  eliminated.  If  we  also  recollect  that,  according  to  an  experiment  of 
Schmidt-^Iltlheim  ^  upon  a  dog,  about  37  per  cent  of  the  given  proteins 
are  absorbed  in  the  first  two  hours  after  the  meal  and  about  59  per  cent 
in  the  course  of  the  first  six  hours,  it  may  then  be  inferred  that  the  in- 
creased elimination  of  nitrogen  after  a  meal  is  due  to  a  catabolization  of 
the  digested  and  assimilated  proteins  of  the  food  not  previously  organized. 
If  it  is  admitted  that  the  catabolized  protein  must  have  been  organized^ 
then  the  greatly  increased  elimination  of  nitrogen  after  a  meal  rich  in 
proteins  supposes  a  far  more  rapid  and  comprehensive  destruction  and 
reconstruction  of  the  tissues  than  has  been  generally  assumed. 

The  extensive  cleavage  of  the  proteins  in  digestion  and  the  repeatedly 
observed  deamidation  of  amino  acids  in  the  animal  body  make  it  prob- 
able that  the  abundant  elimination  of  nitrogen  after  a  diet  rich  in  pro- 
tein is  in  great  part  due  to  a  progressive  demolition  of  the  food  protein  in 


*  Folin,  Amer.  Journ.  of  Physiol.,  13;  Schreuer,  Pfliiger's  Arch.,  110. 

^  Panum,  Nord.  Med.  Arkiv.,  6;  Falck,  see  Hermann's  Handbuch,  6,  Part  I,  107. 
For  further  statements  in  regard  to  the  curve  of  nitrogen  elimination  in  man,  see  Tschen- 
loff,  Korrespond.  Blatt  Schweiz.  Aerzte,  1896;  Rosemann,  Pfliiger's  Arch.,  65,  and 
Veraguth,  Journ.  of  Physiol.,  21;  Schlos.se,  Maly's  Jahre.sber.,  31. 

2  Arch.  f.  (Anat.  u.)  Phy.siol.,  1879. 


NUTRITIVE   VALUE  OF  GELATINE.  745 

digestion  whereby  certain  atomic  complexes  are  more  readily  split  than 
others.  The  abundant  elimination  of  nitrogen  by  the  urine  after  par- 
taking considerable  protein  may  also  depend  in  great  part  upon  these 
nitrogenous  atomic  complexes  which  are  split  off  and  whose  nitrogen  is 
split  off  as  ammonia  and  therefore  cannot  be  used  by  the  body.  The 
abundant  formation  of  ammonia  in  the  cells  of  the  digestive  apparatus 
after  food  rich  in  proteins,  as  observed  by  Nencki  and  Zaleski  *  seem  to 
speak  in  favor  of  this  view. 

In  this  connection  it  must  be  recalled  that,  according  to  the  investi- 
gations of  RiAZANTSEFF,  Substantiated  by  Schepski,  after  partaking  of 
food  an  increased  nitrogen  elimination  depends  in  part  upon  the  increased 
work  of  the  digestive  glands.  The  observations  of  Riazantseff  ^  that 
after  so-called  "apparent  feeding"  an  increased  elimination  of  nitrogen 
occurs  has  not  been  confirmed  by  the  recent  observations  of  Cohxheim 
and  therefore  cannot  be  considered  as  conclusive. 

It  has  been  stated  above  that  other  foods  may  decrease  the  catabolism 
of  proteins.  Gelatine  is  such  a  food.  Gelatine  and  the  gelatine- formers  do 
not  seem  to  be  converted  into  protein  in  the  body,  and  this  last  cannot 
be  entirely  replaced  by  gelatine  in  the  food.  For  example,  if  a  dog  is  fed 
on  gelatine  and  fat,  its  body  sustains  a  loss  of  proteins  even  when  the 
quantity  of  gelatine  is  so  large  that  the  animal,  with  an  amount  of  fat 
and  meat  containing  just  the  same  quantity  of  nitrogen  as  the  gelatine  in 
question,  may  remain  in  nitrogenous  equilibrium.  On  the  other  hand, 
gelatine,  as  Voit,  Panum,  and  Oerum^  have  shown,  has  a  great  value  as 
a  means  of  sparing  the  proteins,  and  it  may  decrease  the  catabolism  of 
proteins  to  a  still  greater  extent  than  fats  and  carbohydrates.  This  is 
apparent  from  the  following  summary  of  Yoit's  experiments  upon  a  dog: 

Food  per  Day.  Flesh. 


Meat.  Gelatine.  Fat.  Sugar.  Catabolized.  Ou  the  Body. 

400  0  200  0                       450                      -50 

400  0                     0  250                     439                      -39 

400  200                  0  0                      356                     +44 

I.  MuNK  *  has  later  arrived  at  similar  results  by  means  of  more  deci- 
sive experiments.  He  found  in  dogs  that  on  a  mixed  diet  which  con- 
tained 3.7  grams  protein  per  kilo  of  body,  of  which  hardly  3.6  grams  was 
catabolized,  nearly  f  could  be  replaced  by  gelatine.     The  same  dog  cata- 

*  Arch,  des  scienc.  biol.  de  St.  Petersbourg  4;  Salaskin,  Zeitschr.  f.  physiol.  Chem. 
25;  Nencki  and  Zaleski,  Arch.  f.  exp.  Path.  u.  Pharm.  37. 

^Arch.  des  scienc.  biol.  de  St.  Petersbourg,  4,  393;  Schepski,  Maly's  Jahresber.,  30; 
Cohnheim,  Zeitschr.  f.  physiol.,  Chem.  46. 

'  Voit,  1.  c,  123;  Panum  and  Oerum,  Nord.  Med.  Arkiv.,  11. 

*Pfluger's  Arch.,  58. 


746  METABOLISM. 

bolized  on  the  second  day  of  starvation  three  times  as  much  protein  as 
with  the  gelatine  feeding.  Munk  states  also  that  gelatine  has  a  much 
greater  sparing  action  on  proteins  than  the  fats  or  the  carbohydrates. 

This  ability  of  gelatine  to  spare  the  proteins  is  explained  by  Voit  by 
the  fact  that  the  gelatine  is  decomposed  instead  of  a  part  of  the  circulat- 
ing proteins,  whereby  a  part  of  this  last  may  be  organized. 

The  recent  investigations  of  Krummacher  and  Kirchmann  show  the 
extent  of  the  sparing  action  of  gelatine  upon  proteins.  The  extent  of 
protein  destruction  during  gelatine  feeding  was  compared  with  the  extent 
■of  protein  catabolism  in  starvation,  and  it  was  found  that  35-37.5  per 
cent  of  the  quantity  of  protein  decomposed  in  starvation  could  be  spared 
by  gelatine.  The  physiological  availability  of  gelatine  was  found  by 
Krummacher  to  be  equal  to  3.88  calories  for  1  gram,  which  corresponds 
to  about  72.4  per  cent  of  the  energy-content  of  the  gelatine.  Kaufmann,^ 
who  experimented  upon  dogs,  found  that  ^  of  the  protein  nitrogen  could 
be  readily  replaced  by  gelatine  nitrogen,  while  in  an  experiment  upon 
himself  with  93  per  cent  gelatine  nitrogen,  4  per  cent  tyrosine  nitrogen, 
2  per  cent  cystin  nitrogen,  and  1  per  cent  tryptophane  nitrogen,  he  found 
instead  of  the  equal  quantity  of  protein  nitrogen  in  the  periods  before  and 
after,  that  the  gelatine  replaced  by  amino  acids  had  nearly  the  same 
physiological  value  as  the  proteins. 

Gelatine  may  also  decrease  somewhat  the  consumption  of  fat,  although 
it  is  of  less  value  in  this  respect  than  the  carbohydrates. 

The  question  of  the  nutritive  value  of  proteoses  (and  peptones)  stands 
in  close  relationship  to  the  nutritive. value  of  the  proteins  and  gelatine. 
The  early  investigations  made  by  Maly,  Plosz  and  Gyergyay,  and 
Adamkiewicz  have  led  to  the  conclusion  that  with  food  which  contains 
no  proteins  besides  peptones  (proteoses)  an  animal  may  not  only  preserve 
its  nitrogenous  equilibrium,  but  its  protein  condition  may  even  increase. 
According  to  recent  and  more  exact  investigations  by  Pollitzer,  Zuntz, 
and  Munk  the  proteoses  have  the  same  nutritive  value  as  proteins,  at 
least  in  short  experiments.  According  to  Pollitzer  this  is  true  for  differ- 
ent proteoses  as  well  as  for  true  peptone;  but  this  does  not  correspond 
with  the  experience  of  Ellinger,-  who  finds  that  the  true  antipeptone 
(gland  peptone)  is  not  able  to  entirely  replace  proteins  or  to  prevent  the 
loss  of  protein  in  the  animal  body.     On  the  contrary,  according  to  him,  it 


'  Knimmacher,  Zeitschr.  f.  Biologic,  42;  Kirchmann,  ibid.,  40;  Kaufmann,  Pfliiger's 
Arch.,  109. 

2  Maly,  Pfliiger's  Arch.,  9;  Plosz  and  Gyergyay,  ibid.,  10;  Adamkiewicz,  "Die  Natur 
und  der  Nahrwerth  des  Peptons"  (Berlin,  1877);  Pollitzer,  Pfliiger's  Arch.,  37,  301; 
Zuntz,  ibid.,  37,  313;  Munk,  Centralbl.  f.  d.  med.  Wissensch.,  1889,  20,  and  Deutsch. 
med.  Wochenschr.,  1889;  Ellinger,  Zeitschr.  f.  Biologic,  33  (literature). 


NUTRITIVE   VALUE   OF   PROTEOSES   AND   PEPTOXES.        747 

has,  like  gelatine,  the  property  of  sparing  proteins.  Voix  long  ago  ex- 
pressed a  similar  view.  According  to  him  the  proteoses  and  peptone  may 
indeed  replace  the  proteins  for  a  short  time,  but  not  permanently;  they 
can  spare  the  proteins,  but  cannot  be  converted  into  proteins.  According 
to  the  researches  of  Blum  ^  the  different  proteoses  have  various  nutritive 
values.  In  his  experiments  the  heteroproteose  from  fibrin  could  not  re- 
place the  proteins  of  the  food,  while  casein  protoproteose  had  this  property. 
The  question  as  to  the  nutritive  value  of  proteoses  and  peptones  has 
turned  in  a  new  direction,  due  to  the  more  recent  views,  as  mentioned  in 
Chapter  IX.  on  the  absorption  of  proteins  where  the  proteins  are  not  ab- 
sorbed chiefly  as  proteoses  and  peptones,  but  as  simpler  cleavage  products. 
From  these  simple  products  as  mentioned  in  a  previous  chapter  (IX  on 
absorption)  a  synthesis  of  protein  can  take  place  in  the  body.  Even  if 
such  a  synthesis  takes  place  and  if  it  were  possible  to  nourish  the  body  for 
a  long  time  with  a  mixture  of  digestion  products  still  it  does  not  follow 
that  proteoses  and  peptones  can  completely  replace  the  proteins  of  the  food. 
The  proteoses  and  peptones  are  formed  by  cleagages.  and  perhaps  certain 
atomic  complexes  are  absent  which  occur  in  the  mixture  of  cleavage  pro- 
ducts and  which  are  necessary  for  a  regeneration  of  special  protein  bodies. 
We  have  a  number  of  investigations-  upon  the  value  of  asparagin.  and 
the  results  are  still  not  conclusive  so  that  quite  positive  deductions  can  be 
drawn  from  them.  The  experiments  upon  herbivora  seem  to  indicate  that 
the  asparagin  has  hardly  any  action  upon  the  deposition  of  protein  while 
it  can  have  an  indirect  protein  sparing  action  and  may  serve  in  producing 
temperature.  The  protein  sparing  action  seems,  at  least  in  part,  to  be 
explained  by  its  excelerating  action  upon  digestion.  In  carnivora  (I. 
Muxk)  and  in  mice  (Voix  and  Politis)  it  was  found  that  asparagin  has 
onh"  a  very  slight,  if  any.  sparing  action  on  the  proteins.  It  is  not  known 
how  it  acts  in  man. 

Metabolisin  on  a  Diet  consisting  of  Protein,  with  Fat  or  Carbohydrates. 
Fat  cannot  arrest  or  prevent  the  catabolism  of  proteifis:  but  it  can  decrease 
it,  and  so  spare  the  proteins.  This  is  apparent  from  the  following  table  of 
VoiT.3     A  is  the  average  for  three  days,  and  B  for  six  days. 

Food.  Flesh. 

, « » ' . 

Meat.  Fat.  Metabolized.        On  the  Boay. 

A 1500  0  1512  -12 

B 1500  150  1474  -^26 

»  Zeitschr.  f.  physiol.  Chem.,  30:  Voit.  I.  c.  394. 

-  Weiske,  Zeitschr.  f.  Biologie.  15  and  17.  and  Centralbl.  f.  d.  med.  Wissensch.,  1890, 
945;  Munk,  Virchow's  Arch..  94  and  9S:  Politis,  Zeitschr.  f.  Biologie.  2S.  See  also 
Mauthner.  ibid.,  2S:  Gabriel,  ibid..  29:  and  Voit.  ibid..  29.  125:  Kellner.  Maly's  Jahresber, 
27.  and  Zeitschr.  f.  Biologie.  39:  Kellner  and  Kohler,  Chem.  Centralbl.  1,  1906.  Vsltz 
Pfliiger's  Arch.  107:  v.  Strusiewicz,  Zeitschr.  f.  Biologie  47. 

^  Voit  in  Hermann's  Handbuch  6,  130. 


748  METABOLISM. 

According  to  Voir  the  adipose  tissue  of  the  body  acts  like  the  food-fat, 
and  the  protein-sparing  effect  of  the  former  may  be  added  to  that  of  the 
latter,  so  that  a  body  rich  in  fat  may  not  only  remain  in  nitrogenous  equi- 
librium, but  may  even  add  to  the  store  of  body  proteins,  ^Yhile  in  a  lean 
body  with  the  same  food  containing  the  same  amount  of  proteins  and  fat 
there  would  be  a  loss  of  proteins.  In  a  body  rich  in  fat  a  greater  quantity 
of  proteins  is  protected  from  metabolism  by  a  certain  quantity  of  fat  than 
in  a  lean  body. 

Because  of  the  sparing  action  of  fats  an  animal  to  whose  food  fat  is 
added  may,  as  is  apparent  from  the  table,  increase  its  store  of  protein 
with  a  quantity  of  meat  which  is  insufficient  to  preserve  nitrogenous  equi- 
librium. 

Like  the  fats  the  carbohydrates  have  a  sparing  action  on  the  proteins. 
By  the  addition  of  carbohydrates  to  the  food  the  carnivora  not  only  re- 
mains in  nitrogenous  equilibrium,  but  the  same  quantity  of  meat  which  in 
itself  is  insufficient  and  which  without  carbohydrates  would  cause  a  loss 
of  weight  in  the  body  may  with  the  addition  of  carbohydrates  produce  a 
deposit  of  proteins.     This  is  apparent  from  the  following  table:  ^ 

Fooil.  Flesh. 


Meat. 

Fat. 

Sugar. 

starch. 

Metabolized. 

On  the  Body. 

500 

250 

558 

-   58 

500 

300 

466 

+   34 

500 

200 

505 

-     5 

800 

250 

745 

+   55 

800 

200 

773 

+   27 

2000 

200-300 

1792 

+  208 

2000 

250 

1883 

+  117 

The  sparing  of  protein  by  carbohydrates  is  greater,  as  shown  by  the 
table,  than  by  fats.  According  to  Voit  the  first  is  on  an  average  0  per 
cent  and  the  other  7  per  cent  of  the  administered  protein  without  a  previ- 
ous addition  of  non-nitrogenous  bodies.  Increasing  quantities  of  carbo- 
hydrates in  the  food  decrease  the  protein  metabolism  more  regularly  and 
constantly  than  increasing  quantities  of  fat.  Atwater  and  Benedict  ^ 
also  found  that  the  carbohydrates  had  a  somewhat  greater  sparing  action 
upon  proteins  than  fats. 

Because  of  this  great  protein-sparing  action  of  carbohydrates  the  her- 
bivora,  which  as  a  rule  partake  of  considerable  quantities  of  carbohydrates, 
assimilate  proteins  readily  (Voit). 

The  greater  protein-sparing  action  of  carbohydrates  as  compared  to 
that  of  the  fats  occurs,  as  shown  by  Laxdergren,^  to  a  still  higher  degree 
with  food  poor  in  nitrogen  or  in  nitrogen  starvation,  in  which  cases  the 

'  Voit,  ibid.,  page  143. 

'  See  Ergebni.sse  der  Phy.siologie  3. 

3  L.  c,  Inaug.-Diss.,  and  Skand.  Arcli.  f.  Physiol.,  14. 


SPARING   ACTION   OF  CARBOHYDRATES  AND   FATS.  749 

carbohydrates  have  double  the  protein-sparing  action  as  compared  to  an 
isodynamic  quantity  of  fat. 

The  protein-sparing  action  of  the  carbohydrates  and  fats  has  generally 
been  studied  by  the  one-sided  feeding  with  one  or  the  other  of  these  two 
groups  of  foodstuffs.  The  question  may  be  raised  whether  the  difference 
observed  between  the  fats  and  carbohydrates  could  not  be  brought  about 
also  by  the  simultaneous  supply  of  carbohydrates  and  fat  in  varying  pro- 
portions. Tallquist  ^  has  made  a  series  of  experiments  on  this  subject. 
In  one  of  the  periods  16.27  grams  N,  44  grams  fat,  and  466  grams  carbo- 
hydrate were  given;  in  a  second.  16.08  grams  X,  140  grams  fat,  and  250 
grams  carbohydrate,  containing  nearly  the  same  number  of  calories, 
namely,  2867  and  2873  calories.  In  both  cases  nearly  a  complete  nitro- 
genous equilibrium  was  reached  and  the  carbohydrate  did  not  spare  more 
protein  than  the  fat.  It  is  therefore  possible  that  the  fat  has  about  the 
same  protein-sparing  action  as  an  isodynamic  amount  of  carbohydrate 
when  the  quantity  of  carbohydrates  does  not  sink  below  a  certain  mini- 
mum, which  is  not  known  for  the  present. 

This  condition  as  well  as  the  extent  of  protein-sparing  action  of  the 
carbohydrates  stands,  according  to  Laxdergrex,^  in  close  relation  to  the 
formation  of  sugar  in  the  body.  The  animal  body  always  needs  sugar, 
and  a  lack  of  carbohydrates  in  the  food  leads  to  a  part  of  the  proteins  being 
used  in  the  sugar  formation.  This  part  can  be  spared  by  carbohydrates 
but  not  by  fats,  from  which,  according  to  Laxdergrex,  the  carbohydrates 
cannot  be  formed.  In  this  lies  also  the  probable  reason  why  the  fats,  on 
being  fed  exclusively  but  not  with  a  sufficient  supply  of  carbohydrates, 
have  a  much  lower  protein-sparing  action  than  the  carbohydrates.  The 
fats  cannot  prevent  the  protein  catabolism  necessary  for  the  formation  of 
sugar  on  a  diet  lacking  in  carbohydrates. 

The  law  as  to  the  increased  protein  catabolism  with  increased  protein 
supply  applies  also  to  food  consisting  of  protein  with  fat  and  carbohydrates. 
In  these  cases  the  body  tries  to  adapt  its  protein  catabolism  to  the  supply; 
and  when  the  daily  calorie-supply  is  completely  covered  by  the  food,  the 
organism  can.  within  wide  limits,  be  in  nitrogenous  equilibrium  with  dif- 
ferent quantities  of  protein. 

The  upper  limit  to  the  possible  protein  catabolism  per  kilo  and  per  day 
has  only  been  determined  for  herbivora.  For  human  beings  it  is  not 
known,  and  its  determination  is  from  a  practical  standpoint  of  secondary 
importance.  What  is  more  important  is  to  ascertain  the  lower  limit,  and 
on  this  subject  we  have  several  experiments  upon  man  as  well  as  upon 


^  Finska  Lakaresallskapets  handl.,  1901.     See  also  Arch.  f.  Hygiene,  41. 
*L.  c,  Inaug.-Diss.     See  also  Skand.  Arch.  f.  Physiol.,  14. 


750  METABOLISM. 

dogs  by  HiRSCHFELD,  KuMAGAWA,  Klemperer,  Munk,  Rosenheim,*  and 
others.  It  follows  from  these  experiments  that  the  lower  limit  of  protein 
needed  for  human  beings  for  a  week  or  less  is  about  30-40  grams  or  0.4- 
0.6  gram  per  kilo  with  a  body  of  average  weight,  v.  Noorden  ^  considers 
0.6  gram  protein  (absorbed  protein)  per  kilo  and  per  day  as  the  lower 
limit.  The  above-mentioned  figures  are  only  valid  for  short  series  of  ex- 
periments; still  there  exist  the  observations  of  E.  Voit  and  Constantinidi  ^ 
on  the  diet  of  a  vegetarian  when  the  protein  condition  was  kept  nearly 
normal  but  not  completely  for  a  long  time  with  about  0.6  gram  of  protein 
per  kilo.  Caspari  *  has  also  made  observations  upon  a  vegetarian  and  for 
a  period  of  14  days  with  an  average  of  0.1  gram  nitrogen  (recalculated  as 
equal  to  0.62  gram  protein)  per  kilo  where  a  nearly  complete  nitrogenous 
equilibrium  was  observed  as  the  average  result. 

According  to  Voit's  normal  figures,  which  will  be  spoken  of  below,  for 
the  nutritive  need  of  man  an  average  working  man  of  about  70  kilos 
weight  requires  on  a  mixed  diet  about  40  calories  per  kilo  (true  calories 
or  net  calories).  In  the  above  experiments  with  food  very  poor  in  protein 
the  demand  for  calories  was  considerably  greater;  as,  for  instance,  in  cer- 
tain cases  it  was  51  (Kumagawa)  or  even  78.5  calories  (Klemperer).  It 
therefore  seems  as  if  the  above  very  low  supply  of  protein  was  only  possible 
with  great  waste  of  non-nitrogenous  food;  but  in  opposition  to  this  it  must 
be  recalled  that  in  Voit  and  Constantinidi  's  experiments  upon  the  veg- 
etarian, who  for  years  was  accustomed  to  a  food  very  poor  in  protein  and 
rich  in  carbohydrate,  the  calories  amounted  to  only  43.7  per  kilo.  In  the  case 
studied  by  Caspari  a  supply  of  41  calories  per  kilo  was  entirely  sufficient. 

SivEN  has  shown  by  experiments  upon  himself  that  the  adult  human 
organism,  at  least  for  a  short  time,  can  be  maintained  in  nitrogenous  equi- 
librium with  a  specially  low  supply  of  nitrogen  without  increasing  the  calo- 
ries in  the  food  above  the  normal.  With  a  supply  of  41-43  calories  per 
kilo  he  remained  in  nitrogenous  equilibrium  for  four  days  with  a  supply 
of  nitrogen  of  0.08  gram  per  kilo  of  body  weight.  Of  the  nitrogen  taken, 
a  part  was  of  a  non-protein  nature  and  the  quantity  of  true  protein  nitro- 
gen was  only  0.045  gram,  corresponding  to  about  0.3  gram  of  protein  per 
kilo  of  body  weight.  That  this  low  limit,  which  by  the  way  only  holds 
for  a  short  time,  has  no  general  validity  follows  from  other  observations. 
Thus  Caspari  ^  also,  in  an  experiment  on  himself,  could  not  attain  com- 


1  See  footnote  4,  psge  738;  also  Munk,  Arch.  f.  (Anat.  u.)  Physiol.,  1891  and  1S96; 
Rosenheim,  ibul.,  1891;  Pfliiger's  .\rch.,  54. 

'  Grundriss  einer  Methodik  der  Stoffwechseluntersuchungen.     Berlin.  1892. 

^  Zeitschr.  f.  Biologie,  25. 

''  Physiologische  Studien  liber  Vegetarismus,  Bonn,  1905. 

^Siven,  Skand.  Arch.  f.  Physiol.,  10  and  11;  Caspari,  Arch.  f.  (Anat.  u.)  PhysioU 
1901. 


DKPOSITIOX   OF   FLESH. 


751 


plete  nitrogenous  equilibrium  on  a  much  greater  nitrogen  supply.  The 
protein  minimum  seems  also  to  be  different  for  various  individuals. 

The  very  important  question  as  to  the  conditions  favoring  the  depo- 
sition of  fat  and  flesh  in  the  body  is  closely  associated  with  \Yhat  has  just 
been  said  in  regard  to  foods  consisting  of  protein  and  non-nitrogenous 
foodstuffs.  In  this  connection  it  must  be  remembered  in  the  first  place 
that  all  fattening  presupposes  an  overfeeding,  i.e.,  a  supply  of  foodstuffs 
which  is  greater  than  that  catabolized  in  the  same  time. 

In  carnivora  a  flesh  deposition  may  take  place  on  the  exclusive  feeding 
with  meat.  This  is  not  generally  large  in  proportion  to  the  quantity  of 
protein  catabolized.  As  shown  by  an  experiment  upon  a  male  cat  by 
Pfluger  ^  this  may  be  so  great  that  the  body  doubles  in  weight  under 
favorable  conditions.  In  man  and  herbivora,  on  the  contrary,  the  demand 
for  calories  may  not  be  covered  by  protein  alone,  and  the  question  as  to 
the  conditions  of  fattening  with  a  mixed  diet  is  of  importance. 

These  conditions  have  also  been  studied  in  carnivora,  and  here,  as 
VoiT  has  shown,  the  relationship  between  protein  and  fat  (and  carbo- 
hydrates) is  of  great  importance.  If  much  fat  is  given  in  proportion  to 
the  protein  of  the  food,  as  with  average  quantities  of  meat  with  consider- 
able addition  of  fat.  then  nitrogenous  equilibrium  is  only  slowly  attained 
and  the  daily  deposit  of  flesh,  though  not  large,  is  quite  constant,  and 
may  become  greater  in  the  course  of  time.  If,  on  the  contrary,  much 
meat  besides  proportionately  little  fat  is  given,  then  the  deposit  of  protein 
with  increased  catabolism  is  smaller  day  b}'  day,  and  nitrogenous  equi- 
librium is  attained  in  a  few  days.  In  spite  of  the  somewhat  larger  deposit 
per  diem,  the  total  flesh  deposit  is  not  considerable  in  these  cases.  The 
following  experiment  of  ^'oIT  may  serve  as  example: 


Number  of 
Days  of  Ex- 
perimentation. 

Food. 

Total 

Deposit  of 

Flesh. 

Daily 

Deposit  of 

Flesh. 

Nitrogenous 

Meat,  Grams. 

Fat,  Grams. 

Equilibrium. 

32 

7 

500 

1800 

250 
250 

1792 
854 

56 
122 

Not  attained 
Attained 

The  greatest  absolute  deposition  of  flesh  in  the  body  was  obtained  in 
these  cases  with  only  500  grams  of  meat  and  250  grams  of  fat,  and  even 
after  32  days  nitrogenous  equilibrium  had  not  occurred.  On  feeding  with 
1800  grams  of  meat  and  250  grams  of  fat  nitrogenous  equilibrium  was 


*  PfliigeV's  Arc-h.,   7". 


752  METABOLISM. 

established  after  seven  days;  and  though  the  deposition  of  fiesh  per  day 
was  greater,  still  the  absolute  deposit  was  not  one  half  as  great  as  in  the 
former  case. 

The  experiments  of  Krug  upon  himself,  under  the  directio.n  of  v. 
NooRDEN,  give  us  information  as  to  the  practicability  of  flesh  deposition 
in  man.  With  abundant  food  (2590  cal.  =44  cal.  per  kilo)  Krug  was 
close  to  nitrogenous  equilibrium  for  six  days.  He  then  increased  the 
nutritive  supply  to  4300  cal.  =71  cal.  per  kilo  for  fifteen  days  by  the  addi- 
tion of  fat  and  carbohydrate,  and  in  this  time  309  grams  of  protein,  corre- 
sponding to  1455  grams  of  muscle,  was  spared.  Of  the  excess  of  admin- 
istered calories  in  this  case  only  5  per  cent  was  used  for  flesh  deposit  and 
95  per  cent  for  fat  deposit.  On  the  other  hand  Bornstein,'  also  experi- 
menting upon  himself,  without  any  considerable  increase  in  calories,  could 
produce,  an  increase  in  his  protein  condition  by  about  100  grams  of 
protein,  corresponding  to  500  grams  of  flesh,  in  the  course  of  fourteen  days 
simply  by  increasing  the  supply  of  protein  (50  grams  of  nutrose  =  sodium 
casein  with  7  grams  N  per  day). 

BoRNSTEiN  arrived  at  still  better  results  in  regard  to  protein  retention 
by  simultaneous  muscle  work,  as  in  these  cases  the  nitrogen  retention 
corresponded  to  a  flesh  deposit  of  800  grams.  The  importance  of  work 
for  the  so-called  protein  deposition  follows  also  from  many  other  obser- 
vations, and  it  is  in  agreement  with  daily  experience  that  a  man  cannot 
be  made  muscle-strong  by  over-feeding  alone.  A  work-hypertrophy 
must  also  be  introduced. 

BoRNSTEiN  and  Schreuer  ^  have  given  further  proof  for  the  possi- 
bility of  a  protein  deposition  in  man  and  animals  (dogs)  and  there  is  no 
doubt  that  the  body  becomes  richer  in  active  cell  masses  after  abundant 
supply  of  protein.  This  increase  seems  still,  according  to  Schreuer,  not 
to  be  continuous,  and  the  question  to  what  extent  the  nitrogen  retention 
in  so-called  protein  overfeeding  in  full-grown  animals  and  man  is  to  be 
considered  as  a  true  flesh  enrichment  i.e.,  a  new  formation  of  living  tissue, 
seems  to  require  further  proof. 

The  conditions  in  young,  growing  individuals  are  different  than  in 
adults.  In  the  first  the  protein  is  necessary  for  the  building  up  of  the 
growing  tissue  and  in  them  an  abundant  true  flesh  deposition  takes  place. 
For  this  protein  fattening  the  amount  of  supply  does  not  take  first  place 
but  rather  the  energy  of  development.  The  growing  body  of  the  nursling 
also  uses  according  to  Rubner  and  Heubner,^  the  protein  of  the  food 

'  Krug,  cited  from  v.  Noorden,  Lehrbuch  der  Pathologie  des  Stoffwechsel.,  2  Aufl. 
557;  Bomstein  Berl.  klin.  Wochenschr.,  1898,  and  Pfluger's  Arch,.  83  and  106. 
'Pfluger's  Arch.,  110. 
^  Zeitschr.  f.  exp.  Path.  u.  Therap.  1. 


ACTION   OF   WATER,   SALTS,   ETC.,  UPOX    METABOLISM.        753 

essentially  to  replace  the  quantity  of  protein  catabolized  and  for  deposi- 
tion. 

It  is  difficult  to  produce  a  permanent  flesh  deposit  in  man  by  overfeed- 
ing alone.  Flesh  deposition  is,  according  to  v.  Xoordex,  a  function  of 
the  specific  energy  of  the  developing  cells  and  the  cell-work  to  a  much 
higher  extent  than  the  excess  of  food.  Therefore  there  is  observed, 
according  to  v.  Xoordex,  abundant  flesh  deposition  (1)  in  each  growing 
body;  (2)  in  those  no  longer  growing  but  whose  body  is  accustomed  to 
increased  work;  (3)  whenever,  by  previous  insufficient  food  or  by  disease, 
the  flesh  condition  of  the  body  has  been  diminished  and  therefore  requires 
abundant  food  to  replace  the  same.  The  deposition  of  flesh  is  in  this  case 
an  expression  of  the  regenerative  energy  of  the  cells. ^ 

The  experiences  of  graziers  show  that  in  food-animals  a  flesh  deposit 
does  not  occur,  or  at  least  is  only  inconsiderable,  on  overfeeding.  The 
individualit}'  and  the  race  of  the  animal  are  of  importance  for  flesh  depo- 
sition. 

As  above  stated  (Chapter  X),  respecting  the  formation  of  fat  in  the 
animal  body,  the  most  essential  condition  for  a  fat  deposition  is  an  over- 
feeding with  non-nitrogenous  foods.  The  extent  of  fat  deposition  is  deter- 
mined by  the  excess  of  calories  administered  over  those  actually  needed. 
If  a  large  part  of  the  calorie-demand  is  covered  by  protein,  then  a  greater 
part  of  the  non-nitrogenous  foodstuffs  simultaneously  ingested  is  spared,  i.e., 
used  for  fat  deposition.  But  as  protein  and  fat  are  expensive  nutritive 
bodies  as  compared  with  carbohydrates,  the  supply  of  greater  quan- 
tities of  carbohydrates  is  important  for  fat  deposition.  The  body  decom- 
poses less  substance  at  rest  than  during  activity.  Bodily  rest,  besides  a 
proper  combination  of  the  three  chief  groups  of  organic  foods,  is  therefore 
also  an  essential  requisite  for  an  abundant  fat  deposit. 

Action  of  Certain  Other  Bodies  on  Metabolism.  Water.  If  a  quantity 
in  excess  of  that  which  is  necessary  is  introduced  into  the  organism,  the 
excess  is  quickly  and  principally  eliminated  with  the  urine.  This  in- 
creased elimination  of  urine  causes  in  fasting  animals  (Voit,  Forster), 
but  not  to  any  appreciable  degree  in  animals  taking  food  (Seegex,  Sal- 
KOW'SKi  and  Muxk,  ^Iayer,  Dubelir  ^),  an  increased  elimination  of  nitro- 
gen. The  reason  for  this  increased  nitrogen  excretion  is  to  be  found  in 
the  fact  that  the  drinking  of  much  water  causes  a  complete  washing  out 
of  the  urea  from  the  tissues.     Another  view,  which  is  defended  by  Voit, 


'  See  also  Svenson,  Zeitschr.  f.  kiln.  Med.,  43. 

^  Voit.  Untersuch,  fiber  den  Einfluss  des  Koch.salzes,  etc.  (Munchen,  1860);  Forster, 
cited  from  Voit  in  Hennann's  Handbuch,  6,  153;  Seegen,  Wien.  Sitzung.sber.,  63;  Sal, 
ko-n-ski  and  Munk,  'S'irchow's  Arch.,  71-;  Mayer,  Zeitschr.  f.  klin.  Med.,  2;  Dubelir- 
Zeitschr.  f.  Biologie,  28. 


75 1  METABOLISM. 

is  that  because  of  the  more  active  current  of  fluids,  after  taking  large 
quantities  of  water  an  increased  metabolism  of  proteins  takes  place.  Yoit 
considers  this  explanation  the  correct  one,  although  he  does  not  deny  that 
by  the  liberal  administration  of  water  a  more  complete  washing  out  of 
the  urea  from  the  tissues  takes  place.  The  views  on  this  question  are 
still  somewhat  contradictory.^ 

When  the  body  has  lost  a  certain  amount  of  water,  then  the  abstinence 
from  water  (in  animals)  is  accompanied  by  a  rise  in  the  protein  metabo- 
lism (Landauer,  Straub-).  In  regard  to  the  action  of  water  on  the 
fomiation  of  fat  and  its  metabolism,  the  view  that  the  free  drinking  of 
water  is  favorable  for  the  deposition  of  fat  seems  to  be  generally  admitted, 
while  the  drinking  of  only  very  little  water  acts  against  its  formation. 

Salts.  The  statements  are  somewhat  contradictory  in  regard  to  the 
action  of  salts,  for  example  sodium  chloride  and  the  neutral  salts,  which 
partly  depends  upon  the  use  of  large  and  varying  amounts  of  salt  in  the 
experiments.  Recent  investigations  of  Straub  and  Rost  ^  have  shown 
that  the  action  of  salts  stands  in  close  relationship  to  their  power  of 
abstracting  water.  Small  amounts  of  salt  which  do  not  produce  diuresis 
have  no  action  on  metabolism.  On  the  contrary,  larger  amounts  which 
bring  about  a  diuresis  which  is  not  compensated  by  the  ingestion  of  water, 
produce  a  rise  in  the  protein  metabolism.  If  the  diuresis  is  compensated 
by  drinking  water,  then  the  protein  metabolism  is  not  increased  by  salts, 
but  is  diminished  to  a  slight  degree.  An  increased  nitrogen  excretion 
caused  by  taking  salts  can  be  somewhat  increased  by  the  ingestion  of 
water  and  thus  increasing  the  diuresis,  and  the  action  of  salts  seems  to 
bear  a  close  relationship  to  the  demand  and  supply  of  water. 

Alcohol.  The  question  as  to  how  far  the  alcohol  absorbed  in  the  intes- 
tinal canal  is  burnt  in  the  body,  or  whether  it  leaves  the  body  unchanged 
by  various  channels,  has  been  the  subject  of  much  discussion.  To  all 
appearances  the  greatest  part  of  the  alcohol  introduced  (95  per  cent  or 
more)  is  burnt  in  the  body  (Stubbotin,  Thudichum,  Bodlander,'Bexe- 
DicENTi  *).  As  the  alcohol  has  a  high  calorific  value  (1  gram  =  7  calories), 
then  the  question  arises  whether  it  acts  sparingl}-  on  other  bodies,  and 
whether  it  is  to  be  considered  as  a  nutritive  substance.  The  older  inves- 
tigations made  to  decide  this  question  have  led  to  no  decisive  result.  The 
thorough  investigations  of  Atwater  and  Benedict,  Zuntz  and  Geppert, 


•See  R.  Neumann,  Arch.  f.  Hygiene,  36;  Heilner,  Zeitschr.  f.  Biologie,  -1:7;  Hawk, 
University  of  Pennsylvania  Med.  Bull.,  xviii. 

^  Landauer,  Maly's  Jahresber.,  24;  Straub,  Zeitschr.  f.  Biologie,  37, 

^W.  Straub,  Zeitschr.  f.  Biologie,  37  and  38;  Rost,  Arbeiten  aus  d.  Kaiserliche 
Gesundheitsamte,  18  (literature).     See  also  Griiber,  Maly's  Jahresber.,  30,  612. 

*Arch.  f.  (Anat.  u.)  Physiol.,  1896,  which  contains  the  literature. 


INFLUENCE   OF  WEIGHT   OF  BODY  AND   AGE.  755 

Bjerre,  Clopatt,  Neumann,  Offer,  Rosemann,^  and  others,  seem  to 
show  positively  that  in  man  alcohol  can  diminish  the  consumption  not 
only  of  fat  and  carbohydrates,  but  also  the  proteins,  although  at  first;, 
due  to  its  poisonous  properties,  it  may  increase  the  protein  metabolismi 
for  a  short  time.  The  nutritive  value  of  alcohol  can  only  be  of  speciaP. 
importance  in  certain  cases,  as  large  amounts  of  alcohol  taken  at  one  time,, 
or  the  continued  use  of  smaller  quantities,  has  an  injurious  action  on  the- 
organism.  Alcohol  may  therefore  be  regarded  as  a.  foodstuff  only  in 
exceptional  cases,  and  in  other  respects  must  be  considered  as  an  article^ 
of  luxury. 

Coffee  and  tea  have  no  action  on  the  exchange  of  material  which  can  bfr 
positively  proved,  and  their  importance  lies  chiefly  in  their  action  upon 
the  nervous  system.  It  is  impossible  to  enter  into  the  effect  of  various 
therapeutic  agents  upon  metabolism. 

V.     The  Dependence  of  Metabolism  on  Other  Conditions. 

The  so-called  starvation  requirement  which  was  previously  mentioned,, 
i.e.,  the  extent  of  metabolism  with  absolute  rest  of  body  and  in  activity  of  the- 
intestinal  tract,  serves  best  as  a  starting-point  for  the  study  of  metabolism 
under  various  external  circumstances.  The  metabolism  going  on  under 
these  conditions  leads  in  the  first  place  to  the  production  of  heat,  and  it  is 
only  to  a  subordinate  degree  dependent  upon  the  work  of  the  circulatory^ 
and  respiratory  apparatus  and  the  activity  of  the  glands.  According  to  a 
calculation  by  Zuntz,^  only  10-20  per  cent  of  the  total  calories  of  the-, 
starvation  requirement  belongs  to  the  circulation  and  respiration  work. 

The  magnitude  of  the  starvation  requirement  depends  in  the  first  place- 
upon  the  heat  production  necessary  to  cover  the  loss  of  heat,  and  this  heat 
production  is  in  turn  dependent  upon  the  relationship  between  the  weight 
and  the  surface  of  the  body. 

Weight  of  Body  and  Age.  The  greater  the  mass  of  the  body  the  greater 
the  absolute  consumption  of  material;  while,  on  the  contrary,  other  things 
being  equal,  a  small  individual  of  the  same  species  of  animal  metabolizes 
absolutely  less,  but  relatively  more  as  compared  with  the  unit  of  the 
weight  of  the  body.  It  must  be  remarked  that  the  relation  between  flesh 
and  fat  in  the  body  exerts  an  important  influence.  The  extent  of  th& 
metabolism  is  dependent  upon  the  quantity  of  active  cells,  and  a  very  fat 

*  In  regard  to  the  literature  on  this  subject,  see  the  works  of  O.  Neumann,  Arch.  f;. 
Hygiene,  36  and  41,  and  Rosemann,  Pfliiger's  Arch.,  86  and  94.     A  summary  of  the- 
entire  hterature  upon  alcohol  can  be  formed  in  Abderhalden,  "Bibliographie  der  ges- 
amten  wissenschaftlichen  Literature  iiber  den  Alcohol  und  den  Alcoholismus,"  Berlio 
and  Wien,  1904. 

'  Cited  from  v.  Noorden's  Handbuch.     2  Aufl. 


756  METABOLISM. 

individual  therefore  decomposes  less  substance  per  kilo  than  a  lean  person 
of  the  same  weight.  According  to  Rubner  '  the  importance  of  the  size 
of  the  flesh  or  cell-mass  in  the  body  is  overestimated.  In  his  investiga- 
tions on  two  boys,  one  of  whom  was  corpulent  and  the  other  normally 
developed,  and  on  comparing  the  food-need  with  that  found  by  Camerer 
for  boys  of  the  same  weight,  Rubner  came  to  the  result  that  the  exchange 
of  force  in  the  corpulent  boy  almost  completely  corresponded  with  that 
in  the  non-corpulent  boy  of  the  same  weight.  B}'  approximately  esti- 
mating the  quantity  of  fat  in  the  body  Rubner  was  also  able,  from  the 
protein  condition,  to  compare  the  calculated  exchange  of  energy  with 
that  actually  found.  The  exchange  per  kilo  amounted  to  52  calories  in 
the  lean  and  43.6  cal.  in  the  fat  boy,  while,  if  the  protein  condition  was  a 
measure,  one  would  expect  an  exchange  of  calories  of  only  35  cal.  for  the 
fat  person.  We  cannot  therefore  admit  of  a  diminished  activity  of  the 
cell-mass  in  the  fat  boy,  but  rather  an  increased  activity.  According  to 
Rubner  it  is  not  the  flesh-mass  (protein  mass)  alone,  but  its  variable 
functional  changes,  which  determines  the  extent  of  decomposition.  In 
women,  who  generally  have  less  body  weight  and  a  greater  quantity  of 
fat  than  men.  the  metabolism  in  general  is  smaller,  and  the  latter  is  ordi- 
narily about  four  fifths  that  of  men. 

The  question  as  to  what  extent  gender  specially  influences  metabolism 
remains  to  be  investigated.  Tigerstedt  and  Sonden  ^  found  that  in  the 
young  the  carbon-dioxide  elimination,  per  kilo  of  body  weight  as  well  as 
per  square  meter  of  body  surface,  was  considerably  greater  in  males  than 
in  females  of  the  same  age  and  the  same  weight  of  body.  This  difference 
between  the  two  sexes  seems  to  disappear  gradually,  and  at  old  age  it 
is  entirely  absent.^ 

The  essential  reason  wh}'  small  animals  catabolize  relatively  more 
.substance  than  large  ones,  when  calculated  per  kilo  body  weight,  is  that 
the  bodies  of  smaller  animals  have  greater  surface  in  proportion  to  their 
mass.  On  this  account  the  loss  of  heat  is  greater,  which  causes  increased 
heat  production,  i.e.,  a  more  active  metabolism.  This  is  also  the  reason 
why  young  individuals  of  the  same  kind  show  a  relatively  greater  meta- 
bolism than  older  ones.  If  the  heat  production  and  carbon-dioxide 
elimination  is  calculated  on  the  unit  of  surface  of  the  body,  we  find,  on 
the  contrary,  as  the  experiments  of  Rubner,  Richet,^  and  others  show, 

'  Beitrage  zur  Ernahrung  im  Knabenalter,  etc.     Berlin,  1902. 

2  Skand.  Arch.  f.  Physiol,  6. 

^  In  regard  to  metabolism  and  its  relationship  to  the  phases  of  sexual  life  and  esi>e- 
cially  under  the  influence  of  menstruation  and  pregnancy,  see  the  investigations  of  A. 
Ver  Eecke  (Bull.  acad.  roy.  de  m6d.  de  Belgique,  1897  and  1901,  and  Maly's  Jahresber., 
SO  and  31). 

*  Rubner,  Zeitschr.  f.  Biologic,  19  and  21;  Richet,  Arch,  de  Physiol,  5.  (2). 


INFLUENCE   OF   AGE.  757 

that  they  vary  only  sHghtly  from  a  certain  average  in  individuals  of  differ- 
ent weights. 

According  to  Rubner's  rule  as  to  the  influence  of  the  surface,  which 
has  been  recently  formulated  by  E.  Voit,  the  need  of  energy  in  homoio- 
thermic  animals  is  influenced  by  the  development  of  their  surface  when 
their  body  is  given  rest,  medium  surrounding  temperature,  and  relatively 
equal  protein  condition.  This  rule  not  only  applies  to  adult  human  beings 
but  also  to  children  and  growing  individuals  (Rubner,  Oppenheimkr). 
The  surface  is  the  essential  factor  in  determining  the  extent  of  exchange 
of  energy.  In  order  to  show  this  we  will  give  here,  from  a  work  of  Rub- 
ner,' the  figures  representing  the  quantity  of  heat  in  calories  for  1  square 
meter  of  surface  for  twenty-four  hours. 

Adult,  medium  diet,  rest 1189  Calories. 

Adult,  medium  diet,  work 1399        " 

Suckling 1221        " 

Child  with  medium  diet 1447        " 

Aged  men  and  women 1099        " 

Women 1004        " 

The  variation  in  the  calorific  values  ^  found  by  many  investigators, 
which  is  sometimes  not  very  small,  speaks  for  the  fact  that  the  surface 
rule  is  not  alone  decisive  for  the  exchange  of  material  in  resting  animals. 
Still  it  is  generally  considered  that  it  is  of  the  greatest  importance  for 
metabolism. 

The  more  active  metabolism  in  young  individuals  is  apparent  when 
we  measure  the  gaseous  exchange  as  well  as  the  excretion  of  nitrogen. 
As  example  of  the  elimination  of  urea  in  children  the  following  results  of 
Camerer  ^  are  of  value: 

Age.  Weight  of  Body  in  Kilos,      p^,.  ]^lf  '"  ^'^'f^;  Kiio. 

l^years 10.80  12.10  1.35 

3  "  13.30  11.10  0.90 

5  "  16.20  12.37  0.76 

7  "  18.80  14.05  0.75 

9  "  25.10  17.27  0.69 

12h  "  32.60  17.79  0.54 

15  "  35.70  17.78  0.50 

In  adults  weighing  about  70  kilos,  from  30  to  35  grams  of  urea  per  day 
are  eliminated,  or  0.5  gram  per  kilo.  At  about  fifteen  years  of  age  the 
destruction  of  proteins  per  kilo  is  about  the  same  as  in  adults.  The  rela- 
tively greater  metabolism  of  proteins  in  young  individuals  is  explained 
partly  by  the  fact  that  the  metabolism  of  material  in  general  is  more  active 

*  Rubner,  Ernahrung  im  Knabenalter,  page  45;   E.  Voit,  Zeitschr.  f.  Biologie,  41  ; 
Oppenheimer,  ibid.,  42. 

^  See  Magnus-Levy,  Pfliiger's  Arch.,  55;  Slowtzoff  (u.  Zuntz),  ibid.,  95. 
^  Zeitschr.  f.  Biologie,  16  and  20. 


758  METABOLISM. 

In  young  animals,  and  partly  by  the  fact  that  young  animals  are,  as  a  rule, 
.poorer  in  fat  than  those  full  grown. 

According  to  Tigerstedt  and  Sonden  the  greater  metabolism  in  young 
animals  depends  nevertheless  also  in  part  on  the  fact  that  in  these  indi- 
viduals the  decomposition  in  itself  is  more  active  than  in  older  ones.  The 
period  of  growth  has  a  considerable  influence  on  the  extent  of  metabolism 
(in  man),  and  indeed  the  metabolism,  even  when  calculated  on  the  unit 
H^i  surface  of  body,  is  greater  in  youth  than  in  old  age.  This  view  is 
strongly  disputed  by  Rubner.  He  does  not  deny  that  differences  exist 
^between  young  and  adult  individuals  which  may  be  considered  as  a  devia- 
ttion  from  the  above  rule;  still  these  differences  may,  according  to  Rubner, 
^e  dependent  upon  the  work  performed,  the  food,  and  the  nutritive  condi- 
tion. Magnus-Levy  and  Falk  ^  have  reported  observations  which  sup- 
port the  views  of  Sonden  and  Tigerstedt. 

In  old  age  the  metabolism  is  very  much  reduced;  and  even  when  calcu- 
lated upon  the  square  meter  of  surface  of  body  it  is  lower  than  in  an  indi- 
vidual of  medium  age. 

As  the  metabolism  may  be  kept  at  its  lowest  point  by  absolute  rest  of 
body  and  inactivity  of  the  intestinal  tract,  it  is  manifest  that  work  and 
•the  ingestion  of  food  have  an  important  bearing  on  the  extent  of  metabo- 
lism. 

Rest  and  Work.  During  work  a  greater  quantity  of  chemical  energy  is 
^converted  into  kinetic  energy,  i.e.,  the  metabolism  is  increased  more  or 
Hess  on  account  of  work. 

As  explained  in  a  previous  chapter  (XI),  work,  according  to  the  gener- 
ially  accepted  view,  has  no  material  influence  on  the  excretion  of  nitrogen. 
It  is  nevertheless  true  that  several  investigators  have  observed  in  certain 
''Cases  an  increased  elimination  of  nitrogen;  but  these  observations  have 
been  explained  in  other  ways.  For  instance,  work  may,  when  it  is  con- 
nected with  violent  movements  of  the  body,  easily  cause  dyspnoea,  and 
"this  last,  as  Frankel^  has  shown,  may  occasion  an  increase  in  the  elimi- 
nation of  nitrogen,  since  diminution  of  the  oxygen  supply  increases  the 
protein  metabolism.  In  other  series  of  experiments  the  quantity  of  car- 
■bohydrates  and  fats  in  the  food  was  not  sufficient;  the  supply  of  fat  in  the 
body  Avas  decreased  thereby,  and  the  destruction  of  proteins  was  corre- 
spondingly increased.  Other  conditions,  such  as  the  external  temperature 
and  the  weather,^  thirst,  and  drinking  of  water,  can  also  influence  the 
excretion  of  nitrogen.  According  to  the  generally  accepted  views  muscu- 
lar activity  has  hardly  any  influence  on  the  metabolism  of  proteins. 

'  Tigerstedt  and  Sonden,  1.  c. ;  Rubner,  1.  c. ;  Magnus-Levy,  Arch.  f.  (Anat.  u.)  Physiol., 
1899,  Suppl. 

*  Virchow's  Arch.,  67  and  7L 

■*See  Zuntz  and  Schumburg,  Arch.  f.  (Anat.  u.)  Physiol.,  1895. 


li\FLUEXCE   OF   REST  AND   WORK.  759^ 

On  the  contrary,  work  has  a  very  considerable  influence  on  the  elimina- 
tion of  carbon  dioxide  and  the  consumption  of  ox3^gen.  This  action, 
which  was  first  observed  by  Lavoisier,  has  later  been  confirmed  by 
many  investigators.  Pettenkofer  and  Voit  ^  have  made  investigations 
on  a  full-grown  man  as  to  the  metabolism  of  the  nitrogenous  as  well  as  of 
the  non-nitrogenous  bodies  during  rest  and  work,  partly  while  fasting 
and  partly  on  a  mixed  diet.  The  experiments  were  made  on  a  full-grown 
man  weighing  70  kilos.     The  results  are  contained  in  the  following  table: 

Consamption  of 


Proteins.  Fat.     Carbohydrates.  COj  Eliminated.  O  Consumed. 

^    ,.  (Rest 79  209  ...  716  761 

tasting  ...  •j^Qj.j. .,5  3gQ  jjg^  jQ_^ 

,,.      ,    ..  ,    J  Rest 1.37  72  352  912  8-31 

Mixed  diet  ^  ^Qj.1. ^37  ^73  3-0  1209  980 

In  these  cases  work  did  not  seem  to  have  any  influence  on  the  destruc- 
tion of  proteins,  while  the  gas  exchange  was  considerably  increased. 

ZuNTz  and  his  pupils  ^  have  made  very  important  investigations  into 
the  extent  of  the  exchange  of  gas  as  a  measure  of  metabolism  during  work 
and  caused  by  work.  These  investigations  not  only  show  the  important 
influence  of  muscular  work  on  the  catabolism  of  material,  but  they 
also  indicate  in  a  very  instructive  way  the  relationship  between  the 
extent  of  metabolism  of  material  and  useful  work  of  various  kinds. 
We  can  only  refer  to  these  important  investigations  which  are  of  special 
physiological  interest. 

The  action  of  muscular  work  on  the  gas  exchange  does  not  alone  appear 
with  hard  work.  From  the  researches  of  Speck  and  others  we  learn  that 
even  very  small,  apparently  quite  unessential  movements  may  increase 
the  production  of  carbon  dioxide  to  such  an  extent  that  by  not  observing 
these,  as  in  numerous  older  experiments,  very  considerable  errors  may 
creep  in.  Johansson  ^  has  also  made  experiments  upon  himself,  and 
finds  that  on  the  production  of  as  complete  a  muscular  inactivity  as  pos- 
sible the  ordinary  amount  of  carbon  dioxide  (31.2  grams  per  hour  at  rest 
in  the  ordinary  sense)  may  be  reduced  nearly  one  third,  or  to  an  average 
of  22  grams  per  hour. 


*  Zeitschr.  f.  Biologie,  2. 

^See  the  works  of  Zuntz  and  Lehmann,  Maly's  Jahresber.,  19;  Katzenstein,  Pfliiger's 
Arch.,  49;  Loewy,  ibid.;  Zuntz,  ibid.,  68,  and  especially  the  large  work  "Untersuch 
uber  den  Stoffwechsel  des  Pferdes  bei  Ruhe  und  Arbeit,"  Zuntz  and  Hagemann,  Berlin 
1898,  which  also  contains  a  bibliography.  Zuntz  and  Slowtzoff,  Pfliiger's  Arch.,  95 ; 
Zuntz,  ibid. 

^  Nord.  Med.  Arkiv.  Festband,  1897;  also  Maly's  Jahresber.,  27;  Speck,  "Physiol, 
des  menschl.  Atmens,"  Leipzig,  1892.  ■ 


760  METABOLISM. 

The  quantity  of  carbon  dioxide  eliminated  during  a  working  period  is 
uniformly  greater  than  the  quantity  of  oxygen  taken  up  at  the  same 
time,  and  hence  a  raising  of  the  respiratory  quotient  was  usually  con- 
sidered as  caused  by  work.  This  rise  does  not  seem  to  be  based  upon  the 
character  of  chemical  processes  going  on  during  work,  as  we  have  a  series 
of  experiments  made  by  Zuntz  and  his  collaborators,  Lehmann,  Kat- 
ZENSTEIN  and  Hagemann,^  in  which  the  respiratory  quotient  remained 
almost  wholly  unchanged  in  spite  of  work.  According  to  Loewy  ^  the 
combustion  processes  in  the  animal  body  go  on  in  the  same  way  in  work 
as  in  rest,  and  a  raising  of  the  respiratory  quotient  (irrespective  of  the 
transient  change  in  the  respiratory  mechanism)  takes  place  only  with 
insufficient  supply  of  ox^-gen  to  the  muscles,  as  in  continuous  fatiguing 
work  or  excessive  muscular  activity  for  a  brief  period,  also  with  local  lack 
of  oxygen  caused  by  excessive  work  of  certain  groups  of  muscles.  This 
varying  condition  of  the  respiratory  quotient  has  been  explained  by  Kat- 
zenstein  by  the  statement  that  during  work  two  kinds  of  chemical  pro- 
cesses act  side  by  side.  The  one  depends  upon  the  work  which  is  con- 
nected with  the  production  of  carbon  dioxide  also  in  the  absence  of  free 
oxygen,  while  the  other  brings  about  the  regeneration  which  takes  place 
by  the  taking  up  of  oxygen.  When  these  two  chief  kinds  of  chemical 
processes  make  the  same  progress  the  respiratory  quotient  remains  un- 
changed during  work;  if  by  hard  work  the  decomposition  is  increased  as 
compared  with  the  regeneration,  then  a  raising  of  the  respiratory  quotient 
takes  place.  If,  on  the  contrary,  moderate  work  is  continued  and  per- 
formed in  a  way  so  that  irregularities  and  occasional  changes  in  the  circu- 
lation and  respiration  are  excluded  or  are  without  importance,  then  the 
respiratory  quotient  may  correspondingly  remain  the  same  during  work 
as  in  rest.  Its  extent  is  thereby  in  the  first  place  determined  by  the  nutri- 
tive material  at  its  disposal  (Zuntz  and  his  pupils). 

The  theory  of  Loewy  and  Zuntz,  that  the  raising  of  the  respiratory  quotient 
during  work  is  to  be  explained  by  an  insufficient  supply  of  oxygen,  is  opposed 
by  Laulanie^'  He  has  observed  the  reverse,  namely,  a  diminution  in  the  respira- 
tory quotient  during  continuous  excessive  work,  and  this  is  not  reconcilable  with 
the  above  statements.  According  to  Laulanik,  who  considers  sugar  as  the  source 
of  muscular  energy,  the  rise  in  the  respiratory  quotient  is  due  to  an  increased 
combustion  of  sugar.  The  diminution  of  the  same  he  explains  by  a  re-formation 
of  sugar  from  fat  which  takes  place  at  the  same  time  and  is  accompanied  by  an 
increased  consumption  of  oxygen. 


'  See  footnote  2,  page  7.59. 

^  Pfliiger's  Arch.,  49. 

3  Arch,  do  Physiol.   (.-)),  8,  572. 


INFLUENCE   OF  EXTERNAL  TEMPERATURE.  761 

In  sleep  metabolism  decreases  as  compared  with  that  during  waking, 
and  the  most  essential  reason  for  this  is  the  muscular  inactivity  during 
sleep.  The  investigations  of  Rubner  upon  a  dog,  and  of  Johansson  ^ 
upon  human  beings,  teach  us  that  if  the  muscular  work  is  eliminated  the 
metabolism  during  waking  is  not  greater  than  in  sleep. 

The  action  of  light  also  stands  in  close  connection  with  the  question  of 
the  action  of  muscular  work.  It  seems  positively  proved  that  metabolism 
is  increased  under  the  influence  of  light.  Most  investigators,  such  as 
Speck,  Loeb,  and  Ewald,-  consider  that  this  increase  is  due  to  the  move- 
ments caused  by  the  light  or  an  increased  muscle  tonus.  Fubini  and 
Benidicenti  ^  assume  that  the  increase  in  metabolism  due  to  light  is  inde- 
pendent of  the  movements.  They  base  this  assumption  on  experiments 
made  on  hibernating  animals. 

Mental  activity  does  not  seem  to  have  any  influence  on  metabolism 
according  to  the  means  at  hand  for  studying  this  influence. 

Action  of  the  External  Temperature.  In  cold-blooded  animals  the  pro- 
duction of  carbon  dioxide  increases  and  decreases  with  the  rise  and  fall  of 
the  surrounding  temperature.  In  warm-blooded  animals  this  condition  is 
different.  By  the  investigations  of  Ludwig  and  Sanders-Ezn,  Pflijger 
and  his  pupils,  and  Duke  Charles  Theodore  of  Bavaria  and  others^  it 
has  been  demonstrated  that  in  warm-blooded  animals  the  change  in  the 
external  temperature  has  different  results  according  as  the  animal's  own 
heat  remains  the  same  or  changes.  If  the  temperature  of  the  animal  sinks, 
the  elimination  of  carbon  dioxide  decreases;  if  the  temperature  rises,  the 
elimination  of  COj  increases.  If,  on  the  contrary,  the  temperature  of  the 
body  remains  unchanged,  then  the  elimination  of  carbon  dioxide  increases 
with  a  lower  and  decreases  with  a  higher  external  temperature.  The 
statements  on  this  subject  are  somewhat  disputed  and  cases  have  been, 
observed  ^^•here  in  warm-blooded  animals  the  merabolism  rises  on  cooling 
and  lowering  the  body  temperature,  while  warming  and  raising  the  body 
temperature  produces  a  diminution  (Krarup  ^). 

The  increase  in  metabolism  produced  by  a  lowering  of  the  external 
temperature  is  explained,  according  to  Pfluger  and  Zuntz,  by  the  state- 


1  Rubner,  Ludwig-Festschr.,  1887;  Loewy,  Berl.  klin.  Wochenschr. ,  1891,  434; 
Johansson,  Skand,  Arch.  f.  Pliysiol.,  8. 

*  Speck,  1.  c;  Loeb,  Pfliiger's  Arch,  42;  Evvald,  Journ.  of  Physiol.,  13. 
'  Cited  from  Maly's  Jahresber.,  22,  395. 

*  The  pertinent  Hterature  may  be  found  cited  by  Voit  in  Hermann's  Handbueh,  6, 
and  also  by  Speck,  1.  c. 

'  J.  C.  Krarup,  Den  omgifvende  temperaturs  indflydeke,  etc.,  Inaug.-Diss.  Kjoben- 
havn,  1902.  See  also  Falloise,  Maly's  Jahresber.,  31;  Predteschcnsky,  ibid;  Rubner, 
Arch.  f.  Hygiene,  38. 


762  METABOLISM. 

nient  that  the  low  temperature,  by  exciting  a  reflex  action  on  the  sensitive 
nerves  of  the  skin,  causes  an  increased  metabolism  in  the  muscles  with  an 
increased  production  of  heat,  affecting  the  temperature  of  the  body,  while 
Avith  a  higher  external  temperature  the  reverse  takes  place.  The  experi- 
ments made  upon  animals  are  somewhat  uncertain  for  several  reasons,  but 
the  determinations  of  the  oxygen  absorption,  as  well  as  the  elimination  of 
CO,,  made  by  Speck,  Loewy,  and  Johansson  ^  in  human  beings,  have 
shown  that  cold  does  not  produce  any  essential  increase  in  the  metabolism 
of  man.  The  irritation  caused  by  cold  may  reflexly  cause  a  forced  respi- 
ration with  an  action  on  the  gas  oxchange,  and  weak  reflex  muscular 
movements,  such  as  shivering,  trembling,  etc.,  may  cause  an  insignificant 
increase  in  the  elimination  of  carbon  dioxide;  in  complete  muscularinac- 
tivity  cold  seems  to  cause  no  increased  absorption  of  oxygen  or  increased 
metabolism.  Eykman's^  experiments  upon  inhabitants  of  the  tropics 
also  show  the  same  result,  namely,  that  in  human  beings  no  appreciable 
heat  regulation  occurs. 

A  very  interesting  and  important  question  is  the  action  of  high  altitude 
upon  the  oxidation  processes,  the  economy  of  temperature,  the  protein 
exchange  and  the  general  metabolism.  The  results  of  the  laborious  and 
important  investigations  on  this  subject  may  be  found  in  the  large  work 
of  N.  ZuxTz,  A.  Loewy,  F.  Muller  and  W.  Caspari.^ 

^Metabolism  is  increased  by  the  ingestion  of  food,  and  Zuntz  has  calcu- 
lated that  in  man  the  consumption  of  oxygen  is  raised  on  an  average  15 
per  cent  above  the  amount  during  rest  for  about  six  hours  after  taking 
a  moderately  hearty  meal.  This  increase  in  the  metabolism  is  caused, 
according  to  the  generally  accepted  view,  probably  only  by  the  increased 
work  of  the  digestive  apparatus  on  the  partaking  of  food.  Rjasantseff 
has  shown  that  the  extent  of  nitrogen  elimination  is  proportioned  to  the 
intensity  of  the  digestive  work.  It  also  follows  from  the  works  of  i\L\GNUS- 
Levy,  Koraen  and  Johansson  *  that  the  proteins  and  to  a  lesser  extent 
the  carbohydrates  even  by  themselves  produce  a  rise  in  metabolism  which 
does  not  seem  to  be  true  for  the  fats. 


•  Speck,  1.  c;  Loewy,  Pfluger's  Arch.,  46;  Johansson,  Skand.  Arch,  f.  Physiol.,  7. 
'  Virchow's  Arch.,  133,  and  Pfluger's  Arch.,  64. 

5  "Hohenklima  und  Bei^wanderungen  in  ihrer  Wirkung  auf  den  Menschen,"  Berlin, 
1906. 

*  Zuntz  and  Levy,  "Beitrag  zur  Kenntniss  d.  Verdaulichkeit,  etc.  ,des  Erodes," 
Pfluger's  Arch.,  49;  Magnus-Levy,  ibid.,  55;  Koraen,  Skand.  Arch.  f.  Physiol.,  11, 
Johansson  and  Koraen,  ibid.,  13. 


FOOD   REQUIREMENT  BY   MAX.  763 

VI.  The  Necessity  of  Food  by  Alan  under  Various  Conditions. 

Various  attempts  have  been  made  to  determine  the  daily  quantity  of 
organic  food  needed  by  man.  Certain  investigators  have  calculated  from 
the  total  consumption  of  food  by  a  large  number  of  similarly  fed  indi- 
viduals—  soldiers,  sailors,  laborers,  etc.  —  the  average  quantity  of  food- 
stuffs required  per  head.  Others  have  calculated  the  daily  demand  of 
food  from  the  quantity  of  carbon  and  nitrogen  in  the  excreta  or  calculated 
it  from  the  exchange  of  force  of  the  person  experimented  upon.  Others, 
again,  have  calculated  the  quantity  of  nutritive  material  in  a  diet  by  which 
an  equilibrium  was  maintained  in  the  individual  for  one  or  several  days 
between  the  consumption  and  the  elimination  of  carbon  and  nitrogen. 
Lastly,  still  others  have  quantitatively  determined  during  a  period  of 
several  daj's  the  organic  foodstuffs  consumed  daily  by  persons  of  various 
occupations  who  chose  their  own  food,  b}^  which  they  were  well  nourished 
and  rendered  fully  capable  of  work. 

Among  these  methods  a  few  are  not  quite  free  from  objection,  and 
others  have  not  as  yet  been  tried  on  a  sufficiently  large  scale.  Neverthe- 
less the  experiments  collected  thus  far  serve,  partly  because  of  their  num- 
ber and  partly  because  the  methods  correct  and  control  one  another,  as  a 
good  starting-point  in  determining  the  diet  of  various  classes  and  similar 
questions. 

If  the  quantity  of  foodstuffs  taken  daily  be  converted  into  calories 
produced  during  physiological  combustion,  we  then  obtain  some  idea  of 
the  sum  of  the  chemical  energy  which  under  varying  conditions  is  intro- 
duced into  the  body.  It  must  not  be  forgotten  that  the  food  is  never 
completely  absorbed,  and  that  undigested  or  unabsorbed  residues  are 
always  expelled  from  the  body  with  the  faeces.  The  gross  results  of  calo- 
ries calculated  from  the  food  taken  must  therefore,  according  to  Rubxer, 
be  diminished  by  at  least  8  per  cent.  This  figure  is  true  at  least  when  the 
human  being  partakes  of  a  mixed  diet  of  about  60  per  cent  of  the  proteins 
as  animal  and  about  40  per  cent  of  the  proteins  as  vegetable  foodstuffs. 
With  more  one-sided  vegetable  food,  especially  when  this  is  rich  in  undi- 
gestible  cellulose,  a  much  larger  quantity  must  be  subtracted. 

The  following  summary  contains  a  few  examples  of  the  quantity  of 
food  which  is  consumed  by  individuals  of  various  classes  of  people  under 
different  conditions.  In  the  last  column  we  also  find  the  quantity  of  liv- 
ing force  which  corresponds  to  the  quantity  of  food  in  question,  calculated 
as  calories,  with  the  above-stated  correction.  The  calories  are  therefore 
net  results,  while  the  figures  for  the  nutritive  bodies  are  gross  results. 


764 


METABOLISM. 


Proteins.  Fat.     ^|rat*S^'    Calories.     Authority. 

Soldier  during  peace  ...  .  119  40  529  2784  Playfair.* 

"      light  service 117  35  447  2424  Hildesheim. 

"      in  field 146  46  504  2852 

Laborer    130  40  550  2903  Moleschott. 

Laborer  at  rest 137  72  352  2458  Pettenkofer  and  VoiT. 

Cabinetmaker  (40  years).  131  68  494  2835  Forster.^ 

Young  physician 127  89  362  2602 

134  102  292  2476 

Laborer  (36  years) 133  95  422  2902 

English  smith    176  71  666  3780  Playfair. 

pugihst  288  88  93  2189 

Bavarian  wood-chopper  .  135  208  876  5589  Liebig. 

Laborer  in  Silesia 80  16  552  2518  Meinert.^ 

Seamstress  in  London ..  .  54  29  292  1688  Playfair. 

Swedish  laborer 134  79  485  3019  Hultgren  and  Landergren.* 

Japanese  student 83  14  622  2779  Eijkman.^ 

Japanese  shopman 55  .6  394  1744  Tawara.^ 

We  have  a  very  large  number  of  complete  investigations  upon  the  diet 
of  people  of  different  vocations  in  America  but  they  are  too  extensive  to 
enter  into,  hence  we  must  refer  to  the  original  publications  of  Atwater.*^ 

It  is  evident  that  persons  of  essentially  different  weight  of  body  who 
live  under  unequal  external  conditions  must  need  essentially  different 
food.  It  is  also  to  be  expected  (and  this  is  confirmed  by  the  table)  that 
not  only  the  absolute  quantity  of  food  consumed  by  various  persons,  but 
also  the  relative  proportion  of  the  various  organic  nutritive  substances, 
shows  considerable  variation.  Results  for  the  daily  need  of  human  beings 
in  general  cannot  be  given.  For  certain  classes,  such  as  soldiers,  laborers, 
etc.,  results  may  be  given  which  are  valuable  for  the  calculation  of  the 
daily  rations. 

Based  on  extensive  investigations  and  a  very  wide  experience,  Voit 
has  proposed  the  following  average  quantities  for  the  daily  diet  of  adults. 

Proteins.  Fat.  Carbohydrates.     Calories. 

For  men 118  grams         56  grams         500  grams         2810 

But  it  should  be  remarked  that  these  data  relate  to  a  man  weighing 
70  to  75  kilos  and  who  was  engaged  daily  for  ten  hours  in  not  too 
fatiguing  labor. 

The  quantity  of  food  required  by  a  woman  engaged  in  moderate  work 


'  In  regard  to  the  older  researches  cited  in  this  table  we  refer  the  reader  to  Voit  in 
Hermann's  Handbuch,  6,  519. 

^  Ibid.,  and  Zeitschr.  f.  Biologie,  9. 

^  Armee-und  Volkserniihrung,  Berlin,  1880. 

*  Untensuchung  liber  die  Ernahrung  schwedischer  Arbeiter  bei  frei  gewahlter  Kost 
Stockholm,  1891.     Maly's  Jahresber.,  21. 

^  Cited  from  Kellner  and  Mori  in  Zeitschr.  f.  Biologie,  25. 

»  Report  of  the  Storrs  Agrio.  expt.  Station,  Conn.  1891-1895  and  1896  and  U.  S. 
Report  of  .\griculture,  liull.  53,  1898. 


FOOD   REQUIREMENT   BY   MAX.  7(35 

is  about  four-fifths  that  of  a  laboring  man,  and  we  may  consider  the  f(jl- 
lowing  as  a  daily  diet  with  moderate  work: 

Proteins.  Fat.  Carbohydrates.     Calories. 

For  women C4  grams         45  grams         400  grams         2240 

The  proportion  of  fat  to  carbohydrates  is  here  as  1:  8-9.  Such  a  pro- 
portion occurs  often  in  the  food  of  the  poorer  classes  which  live  chiefly 
upon  the  cheap  and  voluminous  vegetable  food,  while  this  ratio  in  the  food 
of  wealthier  persons  is  1 :  3-4.  It  would  be  desirable  if  in  the  above 
rations  the  fat  was  increased  at  the  expense  of  the  carbohydrates,  but 
unfortunately  on  account  of  the  high  price  of  fat  such  a  modification  can- 
not always  be  made. 

In  examining  the  a]:)Ove  numbers  for  the  daily  rations  it  must  not  be 
forgotten  that  the  figures  for  the  various  foodstuffs  are  gross  results.  They 
consequently  represent  the  quantity  of  these  which  must  be  taken  in,  and 
not  those  which  are  really  absorbed.  The  figures  for  the  calories  are,  on 
the  contrary,  net  results. 

The  various  foods  are,  as  is  well  known,  not  equally  digested  and 
absorbed,  and  in  general  the  vegetable  foods  are  less  completely  consumed 
than  animal  foods.  This  is  especially  true  of  the  proteins.  When,  there- 
fore, VoiT,  as  above  stated,  calculates  the  daily  quantity  of  proteins 
needed  by  a  laborer  as  IIS  grams,  he  starts  M-ith  the  supposition  that  the 
diet  is  a  mixed  animal  and  vegetable  one,  and  also  that  of  the  above  118 
grams  about  105  grams  are  absorbed.  The  results  obtained  by  Pflijger 
and  his  pupils  Borland  and  Bleibtreu  ^  on  the  extent  of  the  metabolism 
of  proteins  in  man  with  an  optional  and  sufficient  diet  correspond  well 
with  the  above  figures,  when  the  unequal  weight  of  body  of  the  various 
persons  experimented  upon  is  sufficiently  considered. 

As  a  rule,  the  more  exclusively  a  vegetable  food  is  employed,  the 
smaller  is  the  quantity  of  proteins  in  the  same.  The  strictly  vegetable 
diet  of  certain  people,  as  that  of  the  Japanese  and  of  the  so-called  vegeta- 
rians, is  therefore  a  proof  that,  if  the  quantity  of  food  be  sufficient,  a  person 
ma}'  exist  on  considerably  smaller  quantities  of  proteins  than  Yoit  sug- 
gests. It  follows  from  the  investigations  of  Hirschfeld,  Kumagawa  and 
Klemperer,  Siven,  and  others  (see  page  750)  that  a  nearly  complete  or 
indeed  a  complete  nitrogenous  equilibrium  may  be  attained  by  the  suffi- 
cient administration  of  non-nitrogenous  nutritive  bodies  with  relatively 
very  small  quantities  of  jDroteins. 

If  we  bear  in  mind  that  the  food  of  people  of  different  countries  varies 
greatl}^  and  that  the  individual  also  takes  essentially  different  nourish- 
ment according  to  the  external  conditions  of  living  and  the  influence  of 
climate,  it  is  not  remarkable  that  a  person  accustomed  to  a  mixed  diet 

1  Bohland,  Pfluger's  Arch.,  36;  Bleibtreu,  ibid.,  38. 


766  METABOLISM. 

can  exist  for  some  time  on  a  strictly  vegetable  diet  deficient  in  proteins. 
No  one  doubts  the  ability  of  man  to  adapt  himself  to  a  heterogeneously 
composed  diet  when  this  is  not  too  difficult  of  digestion  and  is  sufficient 
in  quantity;  also  we  cannot  den}'  that  it  is  possible  for  a  man  to  exist 
also  for  a  long  time  with  smaller  amounts  of  protein  than  Voit  suggests, 
namely  118  grams.  Thus  0.  Xeumaxx  ^  experimented  on  himself  during 
764  days  in  three  series  of  experiments,  and  his  diet  consisted  of  74.2  grams 
protein,  117  grams  fat,  and  213  grams  carbohydrates  =  2367  gross  calo- 
ries, with  a  weight  of  70  kilos  and  with  ordinary  laboratory  work.  These 
figures  cannot  be  compared  with  those  obtained  by  Voit's  worker,  weigh- 
ing 70  kilos,  whose  work  was  harder  than  a  tailor's  and  easier  than  a  black- 
smith's; for  example,  the  work  of  a  mason,  carpenter,  or  cabinet-maker. 
The  very  extensive  investigations  recently  performed  by  Chittexdex  ^ 
on  the  estimation  of  the  extent  of  protein  necessary  are  of  great  interest. 
These  investigations  upon  a  total  of  twenty-six  persons  extended  over  a 
period  of  five  to  twent}' months  and  consisted  of  careful  investigations  and 
observations  upon  the  manner  of  living,  food  taken,  nitrogen  elimination, 
and  the  ability  of  performing  work.  The  different  individuals  were 
divided  into  three  groups.  The  first  consisted  of  five  professional  men 
(four  assistants  and  one  professor).  The  second  group  was  composed  of 
thirteen  soldiers  (of  the  sanitary  corp  of  the  United  States  army)  which 
besides  their  daily  work  were  given  gymnastic  exercises  for  six  months. 
The  third  group  consisted  of  eight  athletic  students  who  were  trained  in 
different  kinds  of  sport. 

In  all  the  persons  experimented  upon  the  original  nitrogen  content 
of  the  food,  which  corresponded  to  Yoit's  value  or  were  somewhat  higher, 
was  gradualh'  reduced  more  or  less.  The  total  calories  supplied  v.ere 
not  increased  above  the  original  value  but  rather  diminished  to  a  reason- 
able extent.  The  bodily  as  well  as  the  mental  abilit}'  was  repeate<-lly 
tested.  As  it  is  not  possible  to  enter  into  the  details  of  the  investigation 
the  following  will  be  sufficient  to  show  the  results.  With  a  diet  corre- 
sponding to  Voit's  values  the  amount  of  urine  nitrogen  per  day  was  16 
grams,  corresponding  to  a  total  protein  catabolism  in  the  body  of  100 
grams  or  1.43  grams  per  kilo.  The  corresponding  results  for  the  aliove 
three  groups  may  be  found  in  the  following  table  where  for  comparison 
Hammarstex  includes  also  the  figures  for  Voit's  diet. 

Urine  Nitrogen.  Catabolized  Protein.  Protein  per  Kilo. 

Min.  Max.  Min.  Max.  Min.  Max. 

Group  1  5.69  8.99  35.6         56.19  0.61  0.S6 

Group  2  7.03  8.39  43.9         52.44  0.74  0.87 

Group  3  7.47  11.06  46.7         69.10  0.75  0.92 

Voit's  figuro«  16                                    100                                  1  43 

'  Arch.  f.  Hygiene.  45. 

-  R.  H.  Chittenden,  Physiological  Economy  in  Nutrition,  New  York,  1904. 


FOOD  REQUIREMENT  BY  MAN.  767 

The  chief  results  from  these  investigations  are  that  on  partaking  of 
amounts  of  protein  much  smaller  than  Voit's  figures,  without  changing 
the  original  supply  of  calories  and  indeed  diminishing  the  same,  the  per- 
sons experimented  upon  remained  not  only  in  nitrogenous  equilibrium, 
but  remained  in  perfect  health  and  were  not  only  able  to  perform  the 
ordinary  work  but  were  indeed  regularly  able  to  perform  much  greater 
work. 

From  these  investigations  which  extended  over  a  long  period  and 
were  carried  on  with  special  care  in  exactitude,  it  cannot  be  denied  that 
man  can  exist  for  a  long  time  with  much  smaller  quantities  of  protein 
than  Voit's  figures  call  for  which  is  also  derived  from  the  experience  of 
vegetarians  and  from  people  living  nearly  entirely  upon  vegetable  food. 
On  the  other  hand  it  must  not  be  forgotten  that  Voit's  figures  represent 
average  results  not  theoretically  necessary  but  which  have  been  shown  to 
be  the  actual  diet  developed  from  habit,  custom,  conditions  of  life  and 
climate,  with  sufficient  nourishment  and  free  selection  for  centuries  in. 
Middle  and  North  Europe.  A  rational  change  in  this  food  requirement 
based  upon  scientific  facts  is  just  as  difficult  to  determine  as  it  is  to  carry 
out  practically.  Certain  standard  figures  for  the  general  needs  of  nutri- 
tion cannot  be  established  because  the  conditions  in  various  countries  are 
different  and  must  necessarily  be  so.  The  numerous  compilations  (of 
At  WATER  and  others  ^)  on  the  diet  of  different  families  in  America  have 
given  the  figures  97-113  grams  protein  for  a  man,  and  the  very  careful 
investigations  of  Hultgren  and  Landergren  have  also  shown  that  the 
laborer  in  Sweden  with  moderate  work  and  an  average  body  weight  of 
70.3  kilos,  with  optional  diet,  partakes  134  grams  protein,  79  grams  fat, 
and  522  grams  carbohydrates.  The  quantity  of  protein  is  here  greater 
than  is  necessary,  according  to  Voit.  On  the  other  hand  Lapicque  ^ 
found  67  grams  protein  for  Abyssinians  and  81  grams  for  ^Malaysians  (per 
body  weight  of  70  kilos),  materially  lower  figures. 

If  we  compare  the  figures  on  page  764  with  the  average  figures  pro- 
posed by  Voit  for  the  daily  diet  of  a  laborer,  it  would  seem  at  the  first 
glance  as  if  the  food  consumed  in  certain  cases  was  considerably  in  excess 
of  the  need,  while  in  other  cases,  as,  for  instance,  that  of  a  seamstress  in 
London,  it  was  entirely  insufficient.  A  positive  conclusion  cannot,  there- 
fore, be  drawn  if  we  do  not  know  the  weight  of  the  body,  as  well  as  the 
labor  performed  by  the  person,  and  also  the  conditions  of  living.     It  is 


'Atwater,  Report  of  the  Storrs  Agric,  Expt.  Station,  Conn.,  1891-1895  and  1896; 
also  Nutrition  investigations  at  the  University  of  Tennessee,  1896  and  1897;  U.  S. 
Dept.  of  Agriculture,  Bull.  53,  1898.  See  also  Atwater  and  Byrant,  ibid.,  Bull.  75; 
Jaffa,  ibid.,  83;  Grindley,  Sammis,  and  others,  ibid.,  91. 

'  Hultgreu  and  Landergren,  1.  c. ;  Lapicque,  Arch,  de  Physiol.  (5),  6. 


768  METABOLISM. 

certainly  tnie  that  the  amount  of  nutriment  required  by  the  body  is  not 
directly  proportional  to  the  body  weight,  for  a  small  body  consumes  rela- 
tively more  substance  than  a  larger  one,  and  varying  quantities  of  fat  may 
also  cause  a  difference;  but  a  large  body,  which  must  maintain  a  greater 
quantity,  consumes  an  absolutely  greater  quantity  of  substance  than  a 
small  one,  and  in  estimating  the  nutritive  need  one  must  also  always  con- 
sider the  weight  of  the  body.  According  to  Voit,  the  diet  for  a  laborer 
with  70  kilos  body  weight  requires  40  calories  for  each  kilo.  Ekholm  ^ 
calculates,  basing  it  upon  his  experiments,  that  for  a  man  weighing  70 
kilos,  busied  with  reading  and  writing,  the  net  calories  are  2450  and  the 
gross  calories  2700,  or  35  and  38.5  calories  per  kilo.  In  the  ordinary  sense 
for  a  resting  man  the  general  food  requirement  is  calculated  in  round  num- 
bers as  30  calories  for  every  kilo.  The  minimum  figure  for  metabolism 
during  sleep  and  in  as  complete  rest  as  possible  has  been  found  by 
SoxDEN,  TiGERSTEDT  and  JoHANSsoN  ^  to  be  24-25  calories. 

As  several  times  stated  above,  the  demands  of  the  body  for  nourish- 
ment vary  with  different  conditions  of  the  body.  Among  these  condi- 
tions two  are  especially  important,  namely,  work  and  rest. 

In  a  previous  chapter,  in  which  muscular  labor  was  spoken  of,  it  was 
seen  that  all  foodstuffs  have  nearly  the  same  power  of  serving  as  a  source 
for  muscular  work,  and  that  the  muscles,  it  seems,  select  that  foodstuff 
Avhich  is  supplied  to  them  in  the  greatest  quantity.  As  a  natural  sequence 
it  is  to  be  expected  that  muscular  activity  requires  indeed  an  increased 
suppl}'  of  foodstuffs,  but  no  essential  change  in  the  relation  of  the  same,  as 
compared  to  rest. 

Still  this  does  not  seem  to  hold  true  in  daily  experience.  It  is  a  well- 
known  fact  that  hard-working  individuals  —  men  and  animals  —  require 
a  greater  quantity  of  proteins  in  the  food  than  less  active  ones.  This  con- 
tradiction is,  however,  only  apparent,  and  it  depends,  as  Voit  has  shown 
upon  the  fact  that  individuals  used  to  violent  work  are  more  muscular. 
For  this  reason  a  person  performing  severe  muscular  labor  requires  food 
containing  a  larger  proportion  of  proteins  than  an  individual  whose 
occupation  demands  less  violent  exertion.  Another  fact  is  that 
the  diet  rich  in  proteins  is  often  concentrated  and  less  bulky,  and 
also  that  in  many  cases  of  training  a  diet  containing  as  little  fat  as 
possible  is  selected. 

If  we  compare  the  results  for  the  needs  of  food  in  work  and  rest  which 
are  obtained  under  conditions  which  can  be  readily  controlled,  it  is  found 
that  the  above  statements  are  confirmed  in  general.     As  example  of  this 

'Skand.  Arch.  f.  Physiol,  11. 

'Sond(5n  and  Tigerstedt,  Skand.  Arch.  f.  Physiol.,  6;  Johansson,  ibid.,  7;  Tigerstedt, 
Nord.  Med.  Arkiv.  Festband,  1897. 


REST  AXD   WORK.  769 

the  following  tables  gives  the  rations  of  soldiers  in  peace  and  in  the  field 
and  the  average  figures  from  the  detailed  data  of  various  countries.^ 

A.   Peace  Ration.  B.  "War  Ration. 


Proteins.  Fat.  Carbohydrates.  Proteins.  Fat.  Carbohydrates. 

Minimum     108  22             504                 126  38             484 

Maximum    165  97             731                 197  95            688 

Mean    130  40             551                 146  59             557 

The  following  figures  for  the  daily  ration  are  obtained  from  the  above 
averages : 

Proteins.  Fat.        Carbohydrates.        Calories. 

In  peace 130       40       551       2900 

In  war  146       59       557       3250 

If  we  calculate  the  fat  in  its  equivalent  quantity  of  starch,  then  the 
relation  of  the  proteins  to  the  non-nitrogenous  foods  is: 

In  peace 1 :4 . 97 

In  war     1:4.79 

The  relation  in  both  cases  is  nearly  the  same.  Similar  results  are 
obtained  when  we  start  with  Voit's  figures  for  a  soldier  in  manoeuvre  A 
(hard  work)  and  B  (strenuous  work)  in  war. 

Proteids.  Fat.        Carbohydrates.       Calories. 

A 135  80  500  3013 

B    145  100  500  3218 

The  relation  here,  when  the  fat  is  recalculated  as  starch,  in  both  cases  is 
the  same,  or  equal  to  1:5. 

If  we  calculate  that  portion  of  the  total  calories  supplied  which  falls 
to  each  group  of  the  foodstuffs,  it  is  found  that  16-19  per  cent  comes  from 
the  protein  in  rest  as  weli  as  with  medium  and  strenuous  work.  For  the 
fat  and  the  carbohydrates  the  variations  are  greater;  the  chief  quantity 
of  calories  comes  from  the  carbohydrates.  Of  the  total  calories  16-30 
per  cent  comes  from  the  fat  and  50-60  per  cent  from  the  carbohydrates. 

The  importance  of  the  food-demand  for  working  individuals  is  shown 
by  the  figures  given  on  page  764  for  a  wood-chopper  in  Bavaria.  A  need 
of  more  than  4000  calories  occurs  only  seldom,  and  with  very  hard  work 
the  demand  may  rise  even  to  7000  calories  (Atwater  and  Bryant, 
Jaffa  ^). 

As  more  work  requires  an  increase  in  the  absolute  quantity  of  food,  so 
the  quantity  of  food  must  be  diminished  when  little  work  is  performed. 

^  Germany,  Austria,  Switzerland,  France,  Italy,  Russia,  and  the  United  States.     It 
is  not  known  by  the  author  whether  these  figures  have  been  changed  in  the  last  few 
years  in  the  various  countries,  and  hence  whether  they  must  be  modified  or  not. 
'See  footnote  1,  page  767, 


770  METABOLISM. 

The  question  as  to  how  far  this  can  be  done  is  of  importance  in  regard  to 
the  diet  in  prisons  and  poorhouses.  We  give  below  the  following  as  ex- 
ample of  such  diets: 

Proteins.  Fat.  Carbohydrates.  Calories. 

Prisoner  (not  working) 87  22  305  1667     Schuster.^ 

Prisoner  (not  working) 85  30  300  1709     Voit, 

Man  in  poorhouse 92  45  332  1985     Forster.^ 

Woman  in  poorhouse 80  49  266  1725 

The  figures  given  by  Voit  are,  he  says,  the  lowest  reported  for  a  non- 
working  prisoner.  He  considers  the  following  as  the  lowest  diet  for  old 
non- working  people: 

Proteins.  Fat.       Carbohydrates.        Calories. 

Men 90  40  350  2200 

Women 80  35  300  1733 

In  calculating  the  daily  diet  it  is  in  most  cases  sufficient  to  ascertain 
how  much  of  the  various  foodstuffs  must  be  administered  to  the  body  in 
order  to  keep  it  in  the  proper  condition  to  perform  the  work  required  of 
it.  In  other  cases  it  may  be  a  question  of  improving  the  nutritive  con- 
dition of  the  body  by  properly  selected  food;  and  there  also  are  cases  in 
which  it  is  desired  to  diminish  the  mass  or  weight  of  the  body  by  an  insuf- 
ficient nutrition.  This  is  especially  the  case  in  obesity,  and  all  the  diet- 
aries proposed  for  this  purpose  are  chiefiy  starvation  cures  which  will  be 
shown  below  from  those  selected,  namely,  Harvey,  Ebstein  and  Oertel's 
cure. 

The  oldest  and  most  gene'rally  known  diet  cure  for  corpulency  is  that  of 
Harvey,  which  is  ordinarily  called  the  Banting  method.  The  principle 
of  this  cure  consists  in  increasing,  as  far  as  possible,  the  consumption  of 
the  accumulated  fat  of  the  body  by  as  limited  a  supply  of  fat  and  carbo- 
hydrates as  practicable  and  a  simultaneously  increased  supply  of  proteins. 
A  second,  called  Ebstein 's  cure,  based  on  the  assumption  (not  correct) 
that  the  fat  of  the  food  is  not  accumulated  in  a  body  rich  in  fat,  but  is 
completely  burnt.  In  this  cure  large  quantities  of  fat  are  therefore 
allowed  in  the  food,  while  the  quantity  of  carbohydrates  is  diminished 
very  materially.  The  third  cure,  called  Oertel's^  cure,  is  based  on  the 
correct  view  that  a  certain  quantity  of  carbohydrates  has  no  greater  influ- 
ence in  the  accumulation  of  fat  than  the  isodynamic  quantities  of  fat.  In 
this  cure,  therefore,  carbohydrates  as  well  as  fat  are  allowed,  provided  the 

*  See  Voit,  Untersuchung  der  Kost.  Miinchen,  1877,  142.  See  also  Hirschfeld 
Maly's  Jahresber..  30. 

"^  See  Voit,  Intersuchung,  der  Kost,  page  186. 

^  Banting,  Letter  on  Corpulence.  London,  1864.  Ebstein,  Die  Fettleibigkeit  und 
ihre  Behandlung.  1882.  Oertel,  Handbuch  der  allg.  Therapie  der  Kreislaufstorungen. 
1884. 


OBESITY  CURES.  771 

total  quantity  of  the  same  is  not  so  great  as  to  hinder  the  decrease  in 
the  fatty  condition.  A  greatly  diminished  supply  of  water  is  also  one  of 
the  features  of  Oertel's  cure,  especially  in  certain  cases.  The  average 
quantity  of  the  various  nutritive  substances  supplied  to  the  body  in  these 
three  cures  is  as  follows,  and  we  give  also  for  comparison  in  the  same  table 
VoiT  's  diet  necessary  for  a  laborer. 

Proteins.         Fat.  Carboliydrates.    ^gJogg^f 

Harvey- Banting's  cure 171  8  75  1083 

Ebstein's  cure 102  85  47  1396 

Oertel's    "    156  22  72  1140 

"(Max) 170  44  114  1573 

Laborer,  according  to  Voit 118  56  500  3055 

If  the  fat  in  all  cases  is  recalculated  in  starch,  then  the  proportion  of 
the  proteins  to  the  carbohydrates  is; 

Harvey-Banting's  cure 100  :     54 

Ebstein's  cure  100  :  240 

Oertel's      " 100  :     80 

"   (Max)   100  :  129 

Laborer 100  :  530 

In  all  these  cures  for  corpulence  the  quantity  of  non-nitrogenous  bodies 
is  diminished  as  compared  with  the  proteins;  but  also  the  total  quantity 
of  food,  as  is  shown  by  the  number  of  calories,  is  considerably  diminished. 

Harvey-Banting's  cure  differs  from  the  others  in  a  relatively  very 
much  greater  quantity  of  proteins,  while  the  total  number  of  calories  in  it 
is  the  smallest.  On  this  account  this  cure  acts  very  quickly;  but  it  is 
therefore  also  more  dangerous  and  more  difficult  to  accomplish.  In  this 
regard  Ebstein's  and  Oertel's  cures  (especially  Oertel's),  having  a 
greater  variation  in  the  selection  of  food,  are  better.  As  the  adipose 
tissue  has  a  protein-sparing  action,  we  have  to  consider  in  using  these 
cures,  especially  Banting's,  that  the  destruction  of  proteins  in  the  body- 
is  not  increased  in  the  adipose  tissue,  and  one  must  therefore  carefully 
watch  the  elimination  of  nitrogen  by  the  urine.  All  diet  cures  for  obesity 
are  moreover,  as  above  stated,  starvation  cures;  and  if  the  daily  quantity 
of  food  required  by  an  adult  man,  represented  as  calories,  is  in  round 
numbers  2500  calories  (according  to  the  average  figures  found  by  Forster 
in  the  case  of  a  physician),  then  one  immediately  sees  what  a  considerable 
part  of  its  own  mass  the  body  must  daily  give  up  in  the  above  cures.  This 
reminds  us  of  the  great  care  necessary  in  employing  them;  each  special 
case  should  be  conducted  with  regard  to  the  individuality,  the  weight  of 
the  body,  the  elimination  of  nitrogen  in  the  urine,  etc.,  etc.,  and  always 
under  strong  control,  and  only  by  a  physician,  never  by  a  layman.  A 
more  detailed  discussion  of  the  many  conditions  which  must  be  considered 
in  these  cases  does  not  enter  the  plan  and  scope  of  this  work. 


AXIMAL  FOODS. 
TABLE  I.— FOODS.  1 


773 


I.  Animal  Foodstuffs. 


1000  Parts  contain 


>,  ■ 

•P"3 


Relationship  of 
1:2:3. 


Meat  without  Boxes. 


Fat  beef  ^ 

Beef  (average  fat  ^) 

Beef  2 

Corned  beef  (average  fat) .  . 

Veal 

Horse,  salted  and  smoked. . 

Smoked  ham 

Pork,  salted  and  smoked  ^. 

Meat  from  hare 

"         "     chicken , 

"         "     partridge 

"         "     wUd  duck 


h.  Meat  with  Bones. 


Fat  beef  ^ 

Beef  (average  fat  ^) 

Beef,  slightly  corned.  .  .  . 
Beef,  thoroughly  corned. 

Mutton,  ver}'  fat 

"        average  fat 

Pork,  fresh,  fat 

"      corned,  fat 

Smoked  ham 


c.  Fishes. 

River  eel,  fresh,  entire 

Salmon,       "  "      

Anchovy,    "  "      

Flounder,    "  " 

River  perch,  fresh,  entire.  .  .  . 
Torsk,  "  "     .  .  .  . 

Pike,  "       ,"•■■■ 

Herring,  salted,  entire 

Ancho\-y,    "  "     

Salmon  (side\  salted 

Kabeljau  fsalted  haddock).  . . 

Codfish  (dried  ling") 

"        (dried  torsk) 

Fish-meal  from  variety  of  Gadus 


183 
196 
190 
218 
190 
318 
255 
100 
233 
195 
253 
246 


156 
167 
175 
190 
135 
160 
100 
120 
200 


89 
121 
128 
145 
100 
86 
82 
140 
116 
200 
246 
532 
665 
736 


166 

98 

120 

115 

80 

65 

365 

660 

11 

93 

14 

31 


141 
83 
93 
100 
332 
160 
460 
540 
300 


220 

67 

39 

14 

2 

1 

1 

140 

43 

108 

4 

5 

10 

7 


11 

18 

18 

117 

13 

125 

100 

40 

12 

11 

14 

12 


9 

15 

85 

100 

8 
10 

5 
60 
70 


6 
10 
11 
11 


6 
100 
107 
132 
178 
106 
59 


640 
688 
672 
550 
717 
492 
280 
130 
744 
701 
719 
711 


544 
585 
480 
430 
437 
520 
365 
200 
340 


352 
469 
489 
580 
440 
455 
461 
280 
334 
460 
472 
257 
116 
170 


150 

150 

167 

180 

88 

150 

70 

80 

90 


333 
333 
333 

250 
450 
450 
4.50 
340 
400 
100 
100 
100 
150 


100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 


100 
100 
100 
100 
100 
100 
100 
100 
100 


100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 

ino 


90 

50 

63 

53 

42 

20 

143 

660 

5 

48 

6 

13 


90 

49 

53 

53 

246 

100 

460 

450 

150 


246 

56 

31 

9 

2 

1 

1 

100 

37 

54 

1 

1 

1 

1 


1  The  resiilts  in  the  following  tableis  are  chiefly  compiled  from  the  simimary  of  Alm^n'  and  of 
Koxic.  "We  here  designate  as  "waste"  that  part  of  the  foods  which  is  lost  in  the  preparation 
or  that  which  is  not  used  by  the  body;  for  instance,  bones,  skin,  egg-shells,  and  the  cellulose 
vegetable  foods. 

2  Meat  such  as  is  ordinarily  sold  in  the  markets  in  Sweden. 

3  Pork,  chiefly  from  the  breast  and  belly,  such  as  occurs  in  the  rations  of  Swedish  soldiers. 


774 


FOOD    TABLES. 
TABLE  I.— YOOT)^— {Continued). 


I.  Animal  Foodstuffs. 


d.  Inner  Organs  (Fresh). 

Brain 

Beef-liver 

Beef-heart 

Heart  and  lungs  of  mutton 

Veal-kidney 

Ox  tongue  (fresh) 

Blood  from  various  animals  (av- 
erage results) 


e.  Other  Animal  Foods. 
Variety  of  pork-sausage  (Mett- 

wurst) 

Same  for  frying 

Butter 

Lard 

Meat  extract 

Cow's  milk  (full) 

"         "     (skimmed) 

Buttermilk 

Cream 

Cheese  (fat) 

"       (poor) 

Whey  cheese  (poor) 

Hen's  egg,  entire 

"        "     without  shell 

Yolk  of  egg 

White  of  egg 


2.  Vegetable  Foodstuffs. 

Wheat  (grains) 

Wheat-flour  (fine) 

"  "      (very  fine) 

W^heat-bran 

Wheat-l)read  (fresh) 

Macaroni 

Rye  (grains) 

Rye-flour 

E,ye-I)read  (dry) 

"         "      (fresh,  coarse) 

"      (fresh,  fine) 

Barley  (grains) 

Scotch  liarley 

Oat  (grains) 

"    (peeled) 

Corn 

Rice  (peeled  for  boiling)   .  . . 

French  beans 

Peas  (yellow  or  green,  dry). 
Flour  from  peas 


1000  Parts  contain 


5  g 
cs.S 

"c  >'- 


116 
196 
184 
1G3 
221 
150 

182 


190 

220 

7 

3 

304 

35 

35 

41 

37 

230 

334 

89 

106 

122 

160 

103 


103 
56 
92 

106 
38 

170 


150 
160 
850 
990 

35 

7 

9 

257 

270 

66 

70 

93 

107 

307 

7 


11 


123 

17 

110 

10 

92 

11 

150 

39 

88 

10 

90 

3 

115 

17 

115 

15 

114 

20 

77 

10 

80 

14 

111 

21 

110 

10 

117 

60 

140 

60 

101 

58 

70 

7 

232 

21 

220 

15 

270 

15 

50 

50 

38 

35 

40 

50 

456 

4 

5 


676 
740 
768 
439 
550 
768 
688 
720 
725 
480 
514 
654 
720 
563 
660 
656 
770 
537 
530 
520 


4 

5 

C 

0) 

11 

770 

17 

720 

10 

714 

10 

721 

13 

728 

10 

670 

9 

807 

50 

610 

55 

565 

15 

119 

7 

175 

217 

7 

873 

7 

901 

7 

905 

6 

665 

60 

400 

50 

500 

56 

329 

8 

654 

10 

756 

13 

520 

8 

875 

18 

1^0 

8 

120 

3 

120 

50 

130 

17 

330 

8 

131 

18 

140 

20 

110 

15 

110 

16 

400 

11 

370 

26 

140 

7 

146 

30 

130 

20 

100 

17 

140 

2 

146 

36 

137 

25 

150 

25 

125 

Relationship  of 
1:2:.3. 


135 


26 
12 

6 
192 

5 

22 

20 

16 

17 

11 

48 

7 

100 

20 

28 

5 

37 

60 

45 


100 
100 
100 
100 
100 
100 

100 


100 
100 
100 
100 

100 
100 
100 
100 
100 
100 
KJO 
100 
100 
100 
100 


100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 


89 
28 
50 
65 
17 
113 

1 


79 

73 

12100 

33000 

100 
20 
22 
695 
117 
19 
79 


192 


VEGETABLE  FOODS  AND  LIQUORS. 
TABLE  L—FOOBS^iContinued). 


1000  Parts  contain 

Relationship  of 

2.  Vegetable  Foodstuffs. 

1 

l| 

2 

3 

"5  ** 

5 -a 

4 

< 

5 

c 

a 
"S 

6 

o 

'S. 

'A 

1 

:2 

•3 

Potatoes 

20 

14 

10 

25 

19 

27 

31 

14 

10 

12 

32 

219 

4 

5 

242 

140 

2 
2 
2 
4 
2 
1 
5 
3 
1 
1 
4 
25 

537 
480 

200 
74 
90 
50 
49 
66 
33 
22 
23 
38 
60 

412 

130 
90 
72 

180 

10 

7 
10 

8 
12 

0 
19 
10 

4 

I 

61 

3 

6 

29 

50 

760 
893 
873 
904 
900 
888 
908 
944 
956 
934 
877 
160 
832 
849 
54 
55 

8 

10 

15 

9 

18 

12 

8 

i 

6 
8 
18 
123 
31 
50 
66 
95 

100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 

10 
14 
20 
16 

11 

4 
16 
21 
10 

8 
12 
12 

222 
343 

1030 

Turnips        

529 

Carrot  (vellow) 

900 

Cauliflower 

200 

Cabbage 

Beans 

258 
244 

Spinach 

106 

Lettuce 

157 

Cucumbers 

230 

Radishes 

317 

Edil)le  mushrooms  (average).  .  . 
Same  dried  in  the  air  (average).. 
Apples  and  pears 

188 

188 

3250 

Various  berries  (average) 

Almonds 

1800 
30 

Cocoa 

129 

TABLE  IL— MALT  LIQUORS. 


1000  Parts  by  Weight  contain 

c 

<a 
"S 

o  o 

o 

A 
O 
o 

< 

-a 
'S 
o 

3 

c 
"S 

o 

P 

.2 
"S 
< 

o 
o 

>, 

O 

J3 
< 

Porter 

871 

887 
885 
911 
903 
881 
916 

945 

2 

2 
2 
2 
3 

54 

28 
32 
35 
40 
47 
25 

22 

76 

55 
58 
72 
59 

7 

15 

7 
8 
4 
6 
5 

7 

13 

3.0 

2.0 
1.5 

1.7 
4.0 

2 
2 

4 

Beer  (Swedish) 

65 
73 

5 

"      (Swedish  export) 

3 

Draught-beer 

10 

7 

13 

31 

47 

?, 

Lager-beer 

?, 

Bock-beer.  ; 

3 

"Weiss-beer 

9, 

Swedish  "Svagdricka" 

23 

3 

776 


FOOD   TABLES. 


TABLE  III —WINES  AND  OTHER  ALCOHOLIC  LIQUORS. 


1000  Parts  by  Weight  contain 

u 

Is 

Alcohol,  Vol. 
Per  Cent. 

1 

M 
3 

g  6 
111 

1^5 

(0 

a 
o 

< 

'cTo  . 

Bordeaux  wine 

883 
803 
776 
801 
808 
795 
774 
791 
790 
479 

94 
115 

90 

94 
120 
170 
164 
1.56 
164 
263 
460 
550 
442-590 

23 

23 

134 

105 
72 
35 
62 
53 
46 

6 

4 
115 
87 
51 
15 
40 
33 
35 
332 

260-475 

5.9 
5.0 
6.0 
6.0 
7.0 
5.0 
4.0 
5.0 
5.0 

1.0 
1.0 
9.0 
6.0 
2.0 
3.0 
4.0 

2.0 
2.0 
1.0 
2.0 
3.0 
5.0 
3.0 
3.0 
4.0 

White  wine  (Rheingau) 

Champagne 

, 

Rhine  wine  (sparkling) 

Tokay 

(■  60-70 

Sherry 

Port  wine 

Madeira 

Marsala 

Swedish  punch 

Brandy        

French  cognac 

778  INDEX   TO  6PECTRUM  PLATE. 


SPECTRUM  PLATE. 

1.  Absorption  spectrum  of  a  solution  of  oxyhcemogldbin. 

2.  Absorption  spectrum  of  a  solution  of  hcemoglobin,  obtained  by  the  action  of  an 

ammoniacal  ferro- tartrate  solution  on  an  oxyhaemoglobin  solution. 

3.  Absorption  spectrum  of  a  faintly  alkaline  solution  of  methcemoglohin. 

4.  Absorption  spectrum  of  a  solution  of  hcematin  in  ether  containing  oxalic  acid. 

5.  Absorption  spectrum  of  an  alkaline  solution  of  hcematin. 

6.  Absorption  spectrum  of  an  alkaline  solution  of  hcemochromogen,  obtained  by  the 

action  of  an  ammoniacal  ferro- tartrate  solution  on  an  alkaline-hsematin  solution. 
7    Absorption  spectrum  of  an  acid  solution  of  urobilin. 

8.  Absorption  spectrum  of  an  alkaline  solution  of  urobilin  after  the  addition  of  a  zinc- 

chloride  solution 

9.  Absorption  spectrum  of  a  solution  of  lutein  (ethereal  extract  of  the  egg-yolk). 


B     C 


E    b 


\s      \s      7      e     i|      IP     *i     is    1^     U    M    *e    /tr    /a    /o    go 

ji:!ri.ii;'iLimvi^''':i:::'iJil.,:|i.:toMtonitUL^h^^^^^^ 


?/      ?»      S3        ?*       ?5 


!      I 


[] 


ct  X? 


8. 


■H 


/? 


-  ,  a 

/? 

T"¥ 

■ 

I 

u 

m 

w 

^^ET?'" 

J. 

» 

'iiint;[;i|iii]:t!1ini|iiiMiiii|ih!|iu|iii|ini|iiiipi|iiii|'^!|Mi,[iiiijii:!|iiiijiii|iL-i^ilili!i:l|:;^ 

't    U      E     7      a     a     ^    ^^    ^?    ^»     «    /o    /ff     Tr    la    n    7o    ?/     ?3    ?3    2V    25 
BCD  E    b  F  G 


GENERAL  INDEX. 


Abietinic  acid,  336 

Abiuret  products,  54,  394,  417 

Absorption,  412-427 

,  action    of    putrefactive   pro- 
cesses in  the  intestine  on, 
405-407 
Absorption  ratio,  218 

of  the  blood  pigments, 
218 
Acceptor,  6 

Acetanilid,  behavior  in  animal  body,  633 
Acethsemin,  212 
Acetic  acid  in  intestinal  contents,  400 

in  gastric  contents,  373,  376 
,  passage  of,  into  urine,  608,  629 
Aceto-acetic  acid,  625,  671 

in  urine,  625,  668 
Acetone,  32,  669, 

in  urine,  668 
Acetonuria,  668,  669 
Acetophenone,  behavior  in  body,  367 
Acetylene,  compound  with  haemoglobin, 

267 
Acetyldichitosamine,  686 
Acetyl  equivalent,  137 
Acetyl  acid  equivalent,  137 
Acetylamino  benzoic  acid,  636 
Acetylparaminophenol,  633 
Achilles  tendon,  composition  of,  429 
Acholia,  pigmentary,  329 
Achromatin,  149 
Achroo-dextrin,  128 
Acid  albuminates,  36 

,  properties,  48,  49 
,  formation  in  peptic  di- 
gestion, 55,  359 
,  absorption  of,  413 
Acid  amines,  behavior  in  the  animal  body, 

630 
Acid  equivalent,  137 
Acid  fermentation  of  urine,  677 
Acid  haemoglobin,  205 
Acid  rigor,  466 
Acids,    organic,  behavior  in  the  animal 

body,  624,  629-631 
Acidity  of  urine,  544,  545 

of  the  gastric  contents,  374 
of  the  muscles,  448,  466,  467,  469, 
Acrite,  114 
Acrolein,  132 
Acrolein  test,  132,  136 
Acroses,  114 
Acrylic  acid  diureid.     See  Uric  acid. 


Actiniochrom,  691 

Adamkiewicz-Hopkins  reaction,  42,  103 
Adelomorphic  cells,  349,  364, 
Adenase,  16,  271,  273 
Adenine,  157,  162,  271,  571,  578 
,  properties,  reaction,  162 
,  in  urine,  162,  578 
Adhesion,   importance  in  blood  coagula- 
tion, 227 
Adipocere,  442 
Adrenalin,  278,  279 

,  relation  to  glycosuria,  298 
Adrenalin-like  bodies,  278 
iEgagropila,  411 
^rotonometric  method,  709 
Age,  influence  on  metabolism,  755-758 
Agglutination,  195 
Agglutines,  26,  195 
Alanine.  26,  33,  54,  84,  305,  462 
Alanylalanine,  396 
Alanylglycine,  55,  396 
Alanylleucine,  396 
Albumin,  36,  45 

,  detection  of,  in  urine,  640,  643 
,  quantitative  estimation  of,  645 
See  also  Proteids. 
Albumins,  36 

,  general  properties,  39,  45,  60 
.     See  also  the  various  albumins. 
Albuminates,  36 

,  properties,  49,  48 
,  ferruginous    albuminate    in 
the  spleen,  272 
Albuminoids,  37,  72-82,  430,  433,  492 
Albumoids.     See  Albuminoids. 
Albuminose,  in  spermatozoa,  498 
Albuminous  bodies.      See  Proteids. 
Albuminuria,  640 

,  alimentary,  413 
Albumoses.     See  Proteoses. 
Alcapton  and  alcaptonuria,  591,  597,  599 
Alcohol.     See  Ethyl  alcohol. 
Alcoholase,  21 

Alcoholic  fermentation.     See  Ethyl  alco- 
hol. 
Aldehydases  of  the  liver,  18 
Aldehydes,  105 

,  behavior  in  the  animal  body, 
631,  636 
Aleuron  grains,  503 
Alexines,  186 
Aldoses,  105 
Alimentary  glycosuria,  299,  420 

779 


780 


GENERAL   INDEX. 


Alizarin,  in  the  urine,  639 

,  administration  of,  438 
Alkali  albuminates,  36,  48,  49 

,  absorption  of,  413 
Alkali  albumose,  49 

Alkali  carbonates,  physiological    impor- 
tance   for    gaseous 
exchange,  698-702 
,  action  on  secretion  of 

gastric  juice,  350 
,  action  on  secretion  of 
pancreatic  juice,  384 
.     See    various    tissues 
and  fluids. 
Alkalies,  relation    to    gaseous    exchange, 
223,  224 
,  diffusible  and  non-diffusible  in 

blood,  224 
,  division  of,  in  blood  corpuscles 

and  plasma,  224,  238 
.    See  also  the  various  fluids  and 

Alkali  phosphates  in  urine,  619-622,  720 
,  occurrence.      See    the 
various     fluids     and 
organs. 
Alkali  proteose,  49 
Alkali  urates,  542,  575 

in  calculi,  680 
in  sediments,  542,  575,  677, 
678 
(Vlkali  earths,  elimination  by  the  intestine, 
619,  626 
in  urine,  619,  620,  626 
in  bones,  436 

insufficient  supply  of,  438, 
439,  727,  738 
Alkaline  fermentation  of  urine,  677 
Alkalinity,  determination  of,  in  blood,  191 
Alkaloids,  action  on  muscles,  466 

,  passage  of,  into  urine,  639 
,  retention  by  the  liver,  280 
Alkyl  sulphide  of  the  skunk,  692 
Allantoin,  properties  and  occurrence,  568, 
573,  583,  584 
in  transudates,  259,  513 
,  formation  from  uric  acid,  568, 
574 
Alloxan,  568 
Alloxuric  bases,  156,  578 
Alloxuric  bodies,  156 
AUoxyproteic  acid,  611,  613 
Almen-Bottger-Nylander's  sugar  test,  116 

655 
Ambergris,  412 
Ambrain,  412 
Amide  nitrogen,  26,  27 
Amino  acids,  88-103 

,  relation  to  formation  of  uric 

acid,  572 
,  relation  to  formation  of  urea, 

5.'')0-553,  630 
,  relation     to      carbohydrate 
metabolism,  305 


Amino  acids,  formation  from  protein  sub- 
stances,  30,   33,   54,  380, 
394,  401,  416 
,  deamidation  of,  305, 462, 550, 

571 
,  passage  of,  in  the  urine,  614, 

675 
,  conjugation  of,  34 
Amino-acetic  acid.    See  Glycocoll. 
Amino-benzoic  acids,  behavior  in  the  ani- 
mal body, 636 
Amino-caproic  acid.   "See  Leucine. 
Amino-cerebrinic  acid  chloride,  486 
Amino-cerebrinic  acid  glucoside,  486 
Amino-cinnamic  acid,  633,  635 
Amino-glutaric  acid.     See  Glutamic  acid. 
Amino-ethyl  sulphonic  acid.    S°e  Taurine. 
Amino-phenyl-acetic  acid,  behavior  in  ani- 
mal body, 634 
Amino-phenyl-propionic    acid,   30,   585 
Amino-phenyl-propionic  acid,  behavior  in 

the  animal  body,  5S5,  633 
Amino-propionic  acid,   84.      See  also  Al- 
anine. 
Amino-pyrotartaric   acid.     See   Glutamic 

acid. 
Amino  sugar,  107.     See  also  Glucosamine. 
Amino-succinic  acid.     See  Aspartic  acid. 
Amino-thiolactic  acid.     See  Cysteine. 
Amino-valerianic  acid,  84 
Amidulin,  126 
Ammonia,  formation  in  autolysis,  380 

,  formation  in  protein  putrefac- 
tion, 401 
,  formation    from   protein   sub- 
stances, 26,  29,  380,  394,  401 
544 

,  formation  in  tryptic  disection, 
394 

,  occurrence  in  blood,  241,  551 
,  occurrence  in  urine,  544,  549, 

623 
,  elimination    after   administra- 
tion of  mineral  acids,  544, 
624,  625 
,  elimination  in  disease,  549 
,  elimination  in  diseases  of  the 

liver,  554 
,  after  extirpation  or  atrophy  of 
the  liver,  554 
Ammonia,  estimation  of,  in  urine,  623,  624 
Ammonium  salts,  relation  to  formation  of 
glycogen,  291 
,  relation  to  formation  of 

urea,  572 
,  relation  to  formation  of 
uric  acid,  551 
Ammonium-magnesium  phosphate  in  uri- 
nary calculi,  678,  682 
Ammonium-magnesium  phosphate  in  in- 
testinal calculi,  411 
Ammonium-magnesium  phosphate  in  uri- 
nary sediment,  679 


GENERAL    INDEX. 


781 


Aimnoniura  sulphate,  method  of  separat 
ing   proteoses,   38. 
51,  60 
,  method  of  separat- 
ing carbohydrates, 
289 
Ammonium  urate   in  urinary  sediments, 
681 

in  urinary  calculi,  677, 
678 
Amniotic  fluid,  512 
Amphicreatine,  457 
Amphopeptone,  51,  57 
Amygdalin,  17 
Amylose,  126 
Amylodextrin,  126,  128 
Amyloid,  36,  69,  431 

,  vegetable,  129 
Amyloid  degeneration,  bile  in,  329 

,  chondroitin  -sul- 
phuric   acid    in . 
liver  in,  431 
Amylolytic  enzymes,   16,  386,  387.     See 

also  various  tissues  and  secretions. 
Amylopectin,  126 
Amylopsin,  387,  388 
Amylum.     See  Starch. 
Ansemia,  pernicious,  246 
Anaerobic  metabolism,  21,  461 
Aniline,    behavior   in    the    animal    body, 

633 
Anisotropous  substance,  447 
Antedonin,  691 
Antialbumate,  359 
Antialbumid,  56,  359 
Antialbumose,  51 

Antienzymes,  210.      See  also  various  en- 
zymes. 
Antifebrine,  relation  to  elimination  of  uro- 
bilin, 604 
Antimony,  passage  of,  into  milk,  539 

,  action   on   the  elimination  of 
nitrogen,  548 
Antipeptone,  51,  54,  57 
Antipyriiie,  relation  to  formation  of  gly- 
cogen, 291 
,  action  on  the  urine,  604,  638, 
639 
Antitoxines,  25 
Antoxyproteic  acid,  611,  612 
Anuria,  in  cholera,  694 
Aorta  elastin,  75,  76 
Apatite  in  bone-earths,  436 
Aqueous  humor,  264 
Arabinose,  108,  112 

,  relation  to  formation  of  gly- 
cogen, 290 
Arabinosimine,  108 
Arabite,  106 
Arachidic  acid,  131,  518 
Arachoidal  fluid,  257 
Arbacin,  62 

Arbutin,  relation   to  formation  of  glyco- 
gen, 291,  592 
,  behavior  in  the  animal  body,  592 


Arginase,  16,  21,  36,  271,  284,  550 

Aj'ginine,  16,  59,  63,  96,  550 

Argon  in  blood,  697 

Arnold's  aceto-acetic  acid  reaction,  672 

Aromatic  compounds,  behavior  in  animal 

body,  632-f339 
Aromatic  substances  in  the  urine,  586 
Arsenic,  in  the  animal  body,  187,  239,  685, 
695 
action    on    the    elimination    of 
nitrogen,  548 
Arsenious  acid,  action  on  peptic  digestion, 

359 
Arseniuretted  hvdrogen,  poisoning  with, 

331-333 
Arterin,  196 

Ascitic  fluids,  259,  262,  263 
Asparagine,  88 

,  relation  to  formation  of  gly- 
cogen, 291 
,  nutritive  value,  747 
Asparaginic  acid.    See  Aspartic  acid. 
Aspartic  acid,  87 

,  relation    to    formation    of 

uric  acid,  572 
,  relation    to    formation    of 

urea,  550 
,  formation  from  protein,  33, 

54,  88 
,  behavior  in  the  organism, 
550,  572,  630 
Asparagus,  odoriferous  bodies  of,  in  the 

urine,  639 
Assimilation  limit,  259,  260 
Ass's  milk,  529 
Atmidalbumin,  52 
Atmidalbumose,  52 
Atmidkeratin,  75 
Atmidkerato.se,  75 

Atropine,  action   of,    elimination  of   uric 
acid,  569 
,  on  the  secretion  of  saliva,  347 
Auto-digestion,  372.     See  Autolysis. 
Auto-intoxication,  25 
Autolysis,  22,  23 

,  substances    retarding    coagula- 
tion produced  in,  234 
See  also   the  various   organs 
and  tissues. 
Auto-oxidation,  3-8 

Bacterial  proteins,  28 

Bacterium  urea?,  677 

Banting  cure,  770 

Beer- vinegar  bacteria,  enzyme  of,  11 

Beeswax,  138 

Bela's  acetone  reaction.  671 

Bence-.Tones  proteid,  645 

Benzaldehyde,  oxidation  of,  5,  6 

,  substituted  aldehydes,  be- 
havior   in    the    animal 
body,  636 
Benzoic  acid,  formation  from  protein  sub- 
stances. 31.  32,  585 
,  passage  of,  into  sweat,  695 


782 


GENERAL  INDEX. 


Benzoic  acid,  behavior  in  the  organism,  3, 
585,  635 
,  occurrence  in  the  urine,  586 
,  substituted    benzoic     acids, 
action  in  body,  635 
Benzene,  31,  76 

,  behavior   in   the   animal   body, 
632,  633 
Benzoyl-amino-acetic  acid.    See  Hippuric 

acid. 
Benzoyl-cystine,  93 
Benzoar-stones,  411 
Bial's  reagent,  667 
Bifurcated  air,  706 
Bile,  307-334 

,  analysis  of,  326,  327 

,  antiseptic  action,  405-407 

,  enzymes  of,  325 

,  in  disease,  329 

,  influence  on  protein  digestion,  358, 

393,  394,  398,  399 
,  on  the  secretion  of  bile,  309 
,  on  the  absorption  of  fat,  398,  405, 

406,  422-425 
,  on  tryptic  digestion,  393,  394,  399 
,  molecular  concentration  of,  326 
,  passage  of  foreign  bodies,  329 
,  occurrence  of,  in  urine,  426,  652 
,  occurrence   of,    in    gastric    contents, 
373,  398,  399 
in  meconium,  410 
,  decomposition  in  the  intestine,  403 
,  chemical  formation  of,  329-333 
Bile-concretions,  333,  334 
Bile-pigments,  320-325 

,  origin  and  formation,  329- 

333 
,  reactions,  321,322,  652,  653 
,  passage  of,  into  urine,  652 
Biliary  fistulae,  307,  406 

,  influence  of,   on  intestinal 

putrefaction,  406 
,  influence  on  the  food    re- 
quirement, 406, 407 
Bile-salts,  310 
Bile-acids,  311,  319 

in  urine  426,  652 
,  detection  of,  319,  652 
,  absorption  of,  426 
,  origin  of,  330,  331 
,  Pettenkofer's  test  lor,  311 
Bile-mucus,  310,  329 
Bilianic  acid,  315 
Bilicyanin,  319,  322,  324 
Bilifulvin,  320 
BUifuscin,  319,  324 
Bilihumin,  319,  324 
Biliphffiin,  320 
Biliprasin,  319,  324 
Bilipurpurin,  324 
BUirubin,  319,  320 

,  relationship  to  blood-pigments, 

210,  215,  319,  331,  333 
,  relationship      to     hsematoidin, 
216.  320,  321 


Bilirubin,  putrefaction  of,  400 

,  occurrence  of,  319 
Biliverdin,  323 

in  fffices,  409 
Biogen  molecule,  4 
Biogens,  4 

Biological     protein     reaction,     186,     413 
Bismuth,  passage  of,  into  milk,  539 
Birotation,  88,  109 
Bitch's  milk,  529,  535 
Biuret,  34,  555 

Biuret  base,  35;  cleavage  of,  395 
Biuret  reaction,  43,  44,  50,  555 
Bladder.     See  Urinary  calculi. 
Bleeding,  248,  298,  697 
Blister  fluid,  265 
Blonds,  milk  of,  534 
Blood,  170-249 

,  general  behavior,  170,  222,  225 
,  analyses,    quantitative,     235-241 
,  analyses,    physico-chemical,    191, 

236 
,  arterial  and  venous,  196,  197.  241, 

697 
,  defibrinated,  172 
,  asphyxiation,  197,  697 
,  quantity  of,    in    the    body,    248 
,  detection,  chemico-legal,  216 
,  distribution  of,  in  the  organs,  249 
,  behavior  in  starvation,  244 
,  comiX)sition  under  varioxis  condi- 
tions, 241-247 
in  gastic  contents,  373 
in  urine,  648,  650 
Blood-casts,  648 
Blood-clot,  172,  225 
Blood  coagulation,  170,  171, 176-178, 225- 

235 
Blood-corpuscles,  white,  220, 221,  226,  227 
,  number    of,    220,    225, 

226,  246, 247 
,  relation  to  coagulation, 

220 
,  red,  192,  193 
,  number    of,    192,    244, 

246 
,  relation    to    high    alti- 
tudes, 245 
,  passage  of,  into   urine, 

648 
,  permeability  of,  195 
,  composition  of,  218, 219 
Blood  gases,  696,  702 
Blood-pigments,  195-221 

in  bile,  329 
in  urine,  648,  649 
,  estimation,  217,  218 
,  regeneration,  216 
Blood-plasma,  172-183 

,  composition   of,   187,   188, 
238-240 
Blood-plates,  220-222,  227,  228 

,  relation    to   coagulation  of 
blood,  227,  231-233 
Blood-sarum,  172,  183-192 


GENERAL  INDEX. 


rss 


Blood-serum,  composition  of,  188-192 
Blood-spots,  217 
Blood  transfusion,  245,  247,  248 
Blueberries,  coloring  matter  of,  in  urine, 

639 
Blue  stentorin,  691 
Boar  spermatozoa,  497 
Boas'  reaction  for  HCl,  374 

for  lactic  acid,  374 
Bones  and  bone  tissues,  434,  440 

in  starvation,  620 
Bone-earths,  436,  437 
Bone  marrow,  172,  437 
Bonellin,  691 
Borneol,  609,  638 
Bottcher's  spermine  crystals,  496 
Bottger-Almen-Nylander's  sugar  test,  94, 

116,  655 
Bowman's  disks,  488 
Brain,  479-489 
Bromadenine,  159 
Bromanil,  32 
Bromhypoxanthine,  159 
Bromides,  beliavior  to  secretion  of  gastric 

juice,  364 
Bromine,  passage  of,  into  saliva,  346,  347 
Bromoform,  from  protein,  32 

,  behavior  in  the  animal  body, 
631 
Bromtoluene,  behavior  in  the  animal  body, 

636 
Brunettes,  milk  of,  534 
Brunner's  glands,  377 
Buccal  mucus,  341 
Buffy  coat,  225 
Bufidin,  692 
Bufotalin,  692 
Bufotenin,  692 
Bufotin,  692 
Bull,  spermatozoa,  497 
Burbot,  spermatozoa,  62 
Bursfe  mucosae,  contents  of,  266 
Butalanine,  81,  382 
Butter-fat,  517,  518 

,  absorption  of,  424 
Butterfly,  pigment  of  wings,  569,  690 
Buttermilk,  528 
Butyl  alcohol,  behavior  in  the  animal  body, 

632 
Butyric  acid,  in  urine,  607 

in  gastric  contents,  376 
in  milk  fat,  517,  518 
Butyric-acid  fermentation,  5,  110,  516 
in  intestine, 
397,  403 
Butjdmercaptan,  692 
Butyrinase,  in  blood,  185 
Byssus,  37,  81 

Cadaver  alkaloids,  24 
Cadaverine,  24,  98,  457 

in  intestine,  676 

in  urine,  615,  676 
Csecum,  solution  of  cellulose  in  the,  397, 
398 


Caffeine,  157 

,  action  on  the  muscles,  466 
,  behavior  in  the  animal  body,  579 
Calcium,  lack  of,  in  food,  438,  439,  737 

,  occurrence,  625.  See  also  various 
tissues  and  fluids. 
Calcium  carbonate  in  urine,  679 

in  urinary  calculi,  682 
in  urinary  sediments, 

678 
in  bones,  436,  439 
in  tartar,  348 
in  otoliths,  494 
Calcium  casein,  441 
Calcium  oxalate  in  urine,  582 

m  urinary  sediments,  678 
In    urinary    calculi,   681, 
682 
Calcium  phosphate,  relation  to  the  coagula- 
tion  of   fibrinogen, 
229 
,  relation  to  the  coag- 
ulation   of    casein, 
520 
,  occurrence  in  the  in- 
testinal concretions, 
410,  411 
in  the  urine,  543,  619, 

620,  625 
in  urinary  sediments, 

679,  680 
in  urinary  calculi,  681 
682 
Calcimn  salts,  elimination,  619,  626 

,  importance  to  coagulation 
of  the   blood,    171,    177, 
229 
,  importance  to  coagulation 

of  milk,  520 
.    See  various  calcium  salts. 
Calcium  sulphate,  in  urinary  sediments,  679 

,  ion  action,  168 
Calculi,  salivary,  348 

,  intestinal,  410-412 
,  urinary.  680-683 
Calories  of  foodstuffs,  723-727 

of  different  rations,  763-771 
Campho-glucuronic    acid,    122,    610,    638 
Camphor,  behavior  in  the  animal  body, 

610,  638 
Cane-sugar.    See  Saccharose. 
Capranica's  reaction  for  guanine,  161 
Capric  acid,  131,  518,  530 
Caproic  acid,  131,  518,  530 
Caprylic  acid,  131,  518 
Caramel,  115,  124 
Carbamino  acetic  acid,  701 
Carbamic  acid,  563 

in  blood,  186,  5,52 
in  urine,  552,  553,  563 
poisonous  action,  552 
Carbamic-acid  ethylester,  563 
Carbazol,  behavior  in  body,  634 
Carboglobulinic  acid,  701 
Carbohaemoglobins,  207 


r84 


GENERAL  INDEX. 


Carbohydrates,  104-130 

,  importance  in  fat  forma- 
tion, 444,  445,  753 
,  importance     in    glycogen 
formation,  290,  292,  293 
,  importance    for   muscular 
activity,  459,  468,  469, 
473,  474 
,  action  on  protein  metabo- 
lism, 738,  747,  748,  749, 
751 
,  action  on  intestinal  putre- 
faction,   415,    406,    589 
,  absorption  of,  419-421 
,  inadequate  supply  of,  739 
See  also  the  various  car- 
bohydrates. 
Carbolic  acid,  action  on  peptic  digestion, 
359 

See  also  Phenol. 
Carbolic  urine,  591 

Carbon,  relation  to  nitrogen  in  the  urine, 
628,  720 
,  calorific  value,  723 
Carbon  dioxide,  assimilation,  1 

in  the  blood,  696-701, 708- 

710,  712 
in  the  blood  in  diabetes, 

701,  702, 
in  the  blood  in  poisoning 
with  mineral  acids,  701 
in  the  intestine,  401,  403 
in  the  lymph,    251,    702 
in  the  stomach,  372 
in  the  muscles  during  rest 
and  activity,  468,  472 
in   the   muscles   in   rigor 

mortis,  466 
in  the  secretions,  702,  703 
in  transudations,  703 
action  on  the  secretion  of 

gastric  juice,  353 
elimination,  dependence  of 
external      temperature 
upon,  761 
elimination  in  rest  and  ac- 
tivity,   468,    472,    759, 
760 
elimination  by  the  skin, 

695 
elimination     in    the     in- 
cubations   of    the    egg, 
510 
elimmation     in     various 
ages,  756-758 
Carbon-dioxide  haemoglobin,  207,  699 
Carbon-monoxide  poisoning,  205, 299,  460, 
548 
action  on  the  formation  of  lactic  acid, 

460 
action  on  the  elimination  of  nitrogen, 

548 
action  on  the  elimination  of  sugar, 
299.  460 
Carbon  monoxide   hajmochromogen,  209 


Carbon-monoxide  methsemoglobin,  207 
Carbon-monoxide  blood  test,  Hoppe-Sey- 

ler's,  206 
Carminic  acid,  690 
Carnic  acid,  457 
Carniferrine  457 
Carnine,  158,  456,  457 
,  in  urine,  578 
Carnitine,  457 
Carnomuscarine,  457 
Carnosme,  454 
Carp,  s{)erma  of,  63,  504 

,  eggs  of,  71 
Cartilage,  69,  431-434 

,  quantity  of  ash,  434 
,  behavior  to  gastic  juice,  360 
,  behavior  to  pancreatic  juice,  395 
Cartilage  gelatine,  433 
Caseanic  acid,  30,  33,  101 
Caseid,  520 
Casein,  36,  47,  100 

,  origin  of,  537 

,  from  woman's  milk,  530 

,  from  cow's  milk,  518 

,  quantitative  estimation  of,  526 

,  absorption  of,  413 

,  behavior  towards  rennin,  362,  520 

,  behavior  to  gastic  juice,  47,  521, 

530,  531 
,  heat  of  combustion,  724 
Caseinokyrin,  58,  59 
Caseinic  acid,  30,  33,  101 
Caseinogen,  521 
Caseoses,  52,  522 

,  relation  to  the  coagulation  of 
blood,  171 
Castor  bean,  25 
Castoreum,  692 
Castorin,  692 
Catalases,  7,  20.     See  also  the  fluids  and 

Catalyzers,  7,  14,  15,  20,  201 

Catheterization    of    the    lungs,    708,    709 

Cat's  milk,  527 

CeH,  animal,  139-169 

Cell  constituents,  primary  and  secondary, 

140 
Cell  fibrinogen,  271 
Cell  globulins,  141,  194 
Cell  membrane,  142,  360 
Cell  nucleus,  149 
Cellulose,  129 

,  fermentation  of,  129,  130,  397, 
404 

,  solution  of,  in  the  caecum,  398 
Cement,  (in  tooth  structure),  440 
Cephalin,  485,  488 
Cephalic  acid,  485 
Cephalopods,  flesh  of,  76,  455,  478 
Cerebrin,  268,  480,  481  483,  484, 

,  in  pus,  268 
Cerebrinin  phosphoric  acid,  486 
Cerebrinic  acid,  486 
Cerebron,  480,  483,  485 
Cerebrosides,  194,  481,  482,  483 


GENERAL  INDEX. 


785 


Cerebrospinal  fluid,  264,  489 

Cerolein.  138 

Cerotic  acid,  138 

Cerumen,  692 

Cervical  ligament,  75,  76,  429 

Cetaceans,  bones  of,  438 

Cetin,  138 

Cetyl  alcohol,  138 

ChakLza,  506 

Charcot's  crj'stals,  247,  496 

'  'Charge  theory,' '  364 

Cheno-taurocholic  acid,  315 

Chief  (adelomorphic)  cells,  349,  364 

Children's  urine,  543,  549,  583 

Chitaminic  acid,  121 

Chitaric  acid,  121 

Chitin,  81,  120,  686 

,  behavior  in  tryptic  digestion,  395 
Chitosamine,  120,  686.     See  also  Gluco- 
samine. 
Chitosan,  687 
Chitose,  121 

Chloral  hvdrate,  behavior  in  the  animal 
body.  609,  632 
,  action  upon  the   secre- 
tion of  bile,  309 
Chloral  secretin,  309 
Chlorates,  poisoning  with,  203,  648 
Chlorazol,  32 
Chlorbenzene,    beha^^or    in    the     animal 

body,  639 
Chlorides,  elimination  by  the  urine,  616- 
619,  694,  695 
,  elimination  by  the  sweat,  695 
,  action  on  protein  metabolism, 

754 
,  insufficient     supply     of,     736 
See  also   various   fluids   and 
tissues. 
Chlorochrome,  282 
Chlorocruorin,  219 

Chloroform,  action  on  the  elimination  of 
chlorides,  615 
,  action  on  the  muscles,  466 
,  action  upon  proteins,  40 
,  behaA-ior  in  the  animal  body, 
631 
Chlorometer,  618 
Clilorophan.  491 
Chlorophyll,  2 

,  relation  to    blood-pigments, 
197,  214 
Clilorosis,  246 

CMorphenylmercapturic  acid,  639 
Clilorrhodinic  acid,  269 
ClJortoluene,  behavior  in  the  animal  bodv, 

636 
Cholagogues,  308 
Cholamine,  315 
Cholalic    acid,    310,    315-318.      See    also 

Cholic  acid. 
Cholanic  acid,  317 
Cholecyanin,  323,  324 
Choleic  acid,  312,  317,  318 


Choleprasin,  319,  324 
Cholepyrrliin,  320 
Cholera,  blood  in,  240 
,  sweat  in,  694 
,  ptomaines  in,  25 
Cholera    bacilli,    behavior    with    gastric 

juice,  371 
Cholestanol,  335 
Cholestenone,  335 
Cholesterilene,  334 
Cholesterin,  334,  335 

in  blood-serum,  183,  194,  220 
in  the  bUe,  310,  326,  327,  328 
in  gaU-stones,  333.  334 
in  the  brain,  480,  487,  488 
in  the  urine,  675,  682 
,  importance  in  thecell,  140, 143 
,  beha\ior  toward  saponin,  337 
Cholest'erin  calculi,  334 
Cholesterin  ester  in  blood-serum,  183 
Cholesterin  fat,  as  protective  fat,  691 
Cholesterin-propionic  ester,  335 
Cholesterinic  acid,  315 
Cholesterone,  334 
Choletelin,  322,  .323 

,  relation  to  urobilin,  603 
Cholic  acids,  315,  316,  335 
Cholic  acid  azide,  312,  315 
Cholic  acid  hydrazide,  312 
Cholic  acid  uretliane,  315 
Choline,  25,  145,  147,  264,  325,  394,  482 
Cholohsematin,  324 
Choloidic  acid,  319 
Cholylic  acid,  316 
Chondrigen,  77,  430 
Chondrin,  81,  269 
Chondrin  balls,  433 
Chondro-albumoid,  433 
Chondroitic  acid,  431 
Choudroitin,  431 

Chondroi tin-sulphuric  acid,  42,  65,  66,  69, 

70, 431 
,  in  urine,  611, 

647 
,  in  kidneys, 
542 
Chondromucoid,  69,  430,  433 
Chondroproteids,  65,  66.  69 

in  the  urine.  647 
Chondrosin    from    chondroitin  -  sulphuric 
acid,  431 
from  sponges,  69 
Chorda  saliva,  340 
Choroid  coat,  493 

,  pigment  of,  688 
Clu-omatin,  149 
Chromhidrosis,  695 
Chromogens  in  urine,  601 

in  suprarenal  capsule.  277 
Chrvsophanic  acid,  action  on  urine,  639 
Chyle.  250-252 
Chyluria,  675 
Chyme,  366 

,  investigation  of,  373-376 


7S6 


GENERAL  INDEX. 


Chymosin,  13,  16,  186,  361,  520 

,  detection  in  gastric  contents, 

373 
,  occurrence    in    the    pancreas, 

386,  395 
.    See  also  Rennin. 
Cilianic  acid,  315 
Cinnamic  acid,  behavior  in  the  animal 

body,  585 
Citric  acid  in  milk,  518,  532 
Clupeine,  63,  64 
Coagulated  proteids,  36,  61 
Coagulation  of  the  blood,  170,  171,  176- 
178, 225-235 
,  intravascular,  234 
of  milk,  362,  373,  396,  515, 

519,  530 
of  muscle-plasma,  448,  452, 
453,  467 
Coagulins,  232,  233 
Coaguloses,  56 
Cobra  poison,  234 
Coccinic  acid,  690 
Coccygeal  glands,  692 
Cochineal,  690 
Cochinillic  acid,  690 
Codfish,  eggs,  504 

,  spermatozoa,  62 
Coefficient,  Haser's,  627 

,  respiratory,  445,  472,  721,  731 
,  dissociation,  190 
,  extinction,  218 
,  urotoxic,  615 
CofTee,  action  on  metabolism,  755 
Collagen,  37,   76-78,   395,   428,  433,   435 
Colloid,  68,  275,  499 
Colloids,  39,  40 
Colloid  corpuscles,  499 
Colloid  cysts,  498 
Colon,  exclusion  of,  427 
Coloring-matters.    See  various  pigments. 
Colostrum,  528,  533 
Colostrum  corpuscles,  528 
Comma    bacillus,    behavior    with    gastric 

juice,  371 
Compound  proteids,  36,  65-72 

.    See  also  the  different  groups 
of  protein  substances. 
Conalbumin,  507 
Conchiolin,  37,  81,  82 
Concentration,    molecular.      See    various 

fluids. 
Concrements.    See  various  calculi. 
Cones  of  the  retina,  pigm-jnt  of,  491 
Conglutin,  calorific  value  of,  724 
Connective  tissues,  428-430 
Copaiva  balsam,  actio.n  on  the  urine,  639 
Copper  in  blood,  187,  239 
in  bile,  310 
in  biliary  calculi,  334 
in  hsemocyanin,  219 
in  protein  substances,  26 
in  turacin,  690 
Cornea,  434,  493 
Cornein,  37,  81,  82 


Cornicrystalline,  82 

Corpora  lutea,  216,  498 

Corpse  wax,  442 

Corpulence,  d  et  cures  for,  779,  771 

Corpus  callosum.,  488 

Corpuscula  amylacea,  486 

Cow's  milk,  515-529 

,  general  behavior,  515,  516 

,  analysis  of,  524-527 

,  anti-putrefactive    action    of, 

405,  588,  589 
,  coagulation  with  rennin,  363, 

373,  516,  530 
,  beliavior  in  the  stomach,  530, 

531 
,  composition  of,  526-528 
Cream,  530 

Creatine,  relation  to  formation  of  urea, 
455,  550 
,  relation  to   muscular   activity, 

470 
,  properties  and  occurrence,  455 
Creatinine,  relation  to  muscular  activity, 
470,  472,  563 
,  properties     and     occurrences, 
563,  564 
zinc  chloride,  564 
Cresol,  30,  401,  588,  589 
Cresol-sulphuric  acid,  588,  589 
Crotonic  acid,  674 
Crude  fibre,  digestion  of,  427 
Cruor,  172 
Crusocreatinine,  457 
Crustaceorubin,  691 

Crusta  inflammatoria  or  phlogistica,  225 
Crystalbumin,  493 
Crystalfibrin,  493 
Crystallins,  491,  492 
Crystalline  lens,  492,  493 
Crystalline  seralbumin,  181 
Crystalloids,  39 
Cimiic  acid,  634,  635 
Cuminuric  acid,  635 
Curd,  442,  520 
Cuorin,  144 
Cyanhaemoglobin,  205 
Cyanhydrines,  106 
Cyanmethtemoglobin,  205 
Cyanocrystalline,  509,  690 
Cyanuric  acid,  555,  568 
Cyanurin,  601 
Cyclopterine,  63 
Cymene,  634 
Cyprinine,  63 
Cysteine,  28,  29,  33,  93,  94 

,  conjugation  in  animal  body,  639 
Cysteinic  acid,  93 
Cystine,  28,  29,  34,  92,  93,  94,  331,  675 

,  occurrence  in  urine,  611,  675,  676 
,  occurrence  in  urinary  sediments, 

680 
,  occurrence  in  urinary  calculi,  682 
,  occurrence  in  sweat.  695 
.  behavior  in  animal  body,  331,675, 
676 


GENERAL  INDEX. 


787 


Cystinuria,  24,  92,  615,  675 
Cvsts,  tapeworm,  265 

,  ovarial,  498,  502 

,  thyroid,  275 

,  mucoid    substances     of,     498-501 
Cvtin,  271 

Cytoglobin,  36,  142,  228,  271 
Cytosine,  152,  156,  165,  166 
Cytotoxines,  186 
Cytozym,  232 

Damaluric  acid,  616 
Damolic  acid,  616 
Defibrinated  blood,  172 
Dehydrochloride  hsemin,  212 
Deliydrocholan,  311 
Dehydrocholic  acid,  315 
Dehydrocholeic  acid,  317 
Dehydrocholesterin,  335 
Delomorphoic  or  parietal  cells,  349,  364 
Denige's  reaction  for  uric  acid,  576 
Denige's-Morner's  tyrosine  test,  91 
Dentin,  437,  440 
Dermoid  cyst,  502 
Dermocerin,  691 
DeiTno  olein,  691 
Desaminoalbuminic  acid,  49 
Desaminoproteic  acids,  31 
Descemet's  membrane,  69,  492 
Desoxycholic  acid,  316 
Deuterocaseoses,  52 
Deuteroelastose,  76 
Deuteroproteose,  52,  60,  644 
Deuterogelatose,  76 
Deuteromyosinose,  79 
Deuterovitellose,  52 
Dextrins,  128 

,  formation  from  starch,  128,  344, 

388 
,  loading  the  stomach  with,  364 
,  occurrence  in  the  gastric  con- 
tents, 367 
,  occurrence  in  muscles,  459 
,  occurrence  in  portal  blood,  242, 
419 
Dextrin-like  substance  in  the  urine,  608 
Dextrose,  114-118 

in  blood,  184,  237,  242,  297-304 
in  urine,  297,  608,  654-665 
in  the  lymph,  250 
in  muscles,  459 
in  the  vitreous  humor,  491 
,  preparation  of,  118 
,  fermentation  of,  20,  115,  657 
,  detection  of,  118,  655-660 
,  reactions  of,  115-117 
,  absorption  of,  419 
,  quantitative    estimation,    600- 
665 
Diabetes  mellitus,  297-306,  654 

,  elimination  of  ammo- 
nia by  the  urine  in, 
625 
,  relationship  of  the  liv- 
er to,  300 


Diabetes  mellitus,  relationship  of  the  pan- 
creas to,  301-303 
,  blood  in,  240,  297 
,  amount    of    sugar    in 

blood  in,  297 
,  urine  in,  543,  628,  654, 

670 
,  CO,  in  the  blood  in,  701 
,  oxybutyric  acid  in  the 

blood  in,  702 
,  oxybutyric  acid  in  the 
urine  in,     625,     673 
Diacetic  acid.     See  aceto-acetic  acid. 
Dialuric  acid,   relationship   to   formation 

of  uric  acid,  573 
Diamide,  poisoning  with,  584 
Diamino  acids,  33,  96-101 
Diamines,  24,  97,  98 

in  the  urine,  615,  676 
in  the  intestinal  contents,  24, 
676 
Diaminoacetic  acid,  97 
Diamino-caproic  acid.     See  Lysine. 
Diaminopropionic  acid-dipeptide,  35 
Diaminotrioxydodecanoic  acid,  101 
Diamino-valerianic  acid,  97.     See  Ornith- 
ine. 
Diastatic  enzymes,  16,  185,  295,  343,  388 

.  See  also  other  enzymes. 
Diastase  in  the  blood,  185 
Diazo  reaction,  Ehrlich's,  612,  613 
Diazobenzol-sulphonic  acid,  reaction  with 

sugar,  117 
Dibenzoylornithin,  97 
Dicalcium  casein,  519 
Dichlorpurme,  157 
Diet  cures,  770,  771 

Diet  for  various  classes  of  people,  763-765 
Digestion,  339-427 
Digestibility  of  food  stuffs,  367-370,  417, 

418,  421,  423,  425 
Diglycyl-glycine,  35 
Dileucyl-glycyl-glycine,  35 
Dileucylcystine,  35 
Dimethylaminobenzaldehyde,      43,     638, 

675 
Dimethylaminobenzoic  acids,  638 
Dimethyltoluidine,  638 
Dimethylketone.     See  Acetone. 
Dioxyacetone,  114 
Dioxybenzenes,  591,  592,  633 
Dioxydiaminosuberic  acid,  33,  101 
Dioxynaphthalene,  633 
Dipalmitylolem,  132 
Dipeptides,  35-37 

,  behavior   with  trypsin,   395, 
396 
Diphtheria  toxins,  action  of  the  gastric 

juice  upon,  371 
Disaccharides,  123 

in  urine,  420,  665,  666 
,  inversion  of,  123,  293,  360, 
378,  419 
as  glycogen  formers,  293, 
294 


788 


GENERAL   INDEX 


Dissociation  degree,  190 
Dissociation  coefficient,  190 
Distearyllecithin,  143 
Distearyipalmitin,  132 
Doeglic  acid,  135 
Dog's  milk,  529,  535 
Dolphin's  milk,  529 
Donne's  pus  test,  651 
Dotterplatchen,  37,  503,  509 
Dropsical  fluid,  262 
Dulcite,  106 

,  relation  to  formation  of  glycogen, 
291 
Dysproteose,  51 
Dyslysins,  319 
Dyspeptone,  359 

Dyspnoea,  action  on  protein  catabolism, 
548,  758 

Ear,  fluids  of,  494 

Earthy  phosphates,  elimination    by    the 
urine,     618,    619, 
625,  626, 
,  absorption  of,  426 
,  solubility    in     fluids 
rich  in  protein,  439 
,  occurrence  in  bone- 
earths,  436-439  _ 
,  occurrence  in  calculi, 
333,  411,  681,  682 
,  occurrence    in    sedi- 
ments,   678,    679 
See   also  different 
earthy  phosphates. 
Ebstein's  diet  cure,  770,  771 
Echinochrom,  219 
Echinococcus  cysts,  cyst  wall,  687 

,  cyst  contents,  265 
Eck's  fistula,  552 
Edestan,  62 
Edestin,  33,  46,  62,  100 

,  absorption  of,  413 
Edible  bird's  nests,  69 
Eel,  flesh  of,  475 
Eel-serum,  171,  234 
Egg,  502 

,  hen's,  502-512 

,  absorption  in  the  intestine,  417 
,  incubation  of,  510-512 
Egg  albumin.    See  Ovalbumin. 
Egg-shell,  73,  323,  509 
Egg  yolk,  502 
Egg-white,  506 

,  albumin  of  the,  507 
Ehrlich's  diazo  reaction.  612,  613 

test  for  bile-pigments,  654 
glucosamine  test,  121 
urine  test,  674 
Elaic  acid,  134 
Elaidic  acid,  135 
Elaidin,  135 

Elastin  proteoses,  76,  77 
Elastin,  37,  75,  76,  100 

,  behavior  with  gastric  juice,  360 
,  behavior  with  tiypsin,  395 


Elephant  bones,  436 
milk,  529 
teeth,  440 
Ellagic  acid,  412 
Embryo  of  the  hen,  development  of,  511, 

512 
Emulsin,  13,  609 
Emvdin,  509 

Enamel  (of  the  teeth),  440 
Encephalin,  482,  484 
Endoenzymes,  22 
Endolymph,  494 

Energy,  potential,  of  foodstuffs,  723-728 
Enterokinase,  379,  383,  384,  386 
Enzymes  in  general,  9-23 
,  zymogens,  16 

.     See   various   enzymes   in   the 
tissues,  organs  and  fluids. 
Epidermis,  73,  685 
Epiguanine,  157,  578,  580 
Epinephrin,  278 
Episarkine,  157,  578,  580 
Erepsin,  153 

,  importance  for  absorption,  380, 
391,  416 
Erucic  acid,  131 

,  absorption  of,  423 
Erythyrite,   relation   to   glycogen  forma- 
tion, 291 
Erythrocytes,     191-196.       See    also    red 

blood-corpuscles. 
Erythrodextrin,  128,  344 
Erythropsin.     See  Visual  purple. 
Esbach's   method  for  estimating  proteid 

in  urine,  645. 
Esters,  cleavage  of,  284,  389 

,  synthesis  of,  390 
Ethal,  138 

Ether,  action  on  the  blood,  193,  195 
,  action  upon  proteins,  40 
,  action  on  the  secretion  of  gastric 

juice,  352 
,  action  on  the  muscles,  466 
Ethereal  sulphuric  acids  in  the  bile,  310, 

325,  327 
Ethereal  sulphuric  acids  in  the  perspira- 
tion, 694 
Ethereal  sulphuric  acids  in  the  urine,  588- 

595,  622,  633,  637 
Ethereal  sulphuric  acids,  synthesis  of,  in 

the  liver,  280 
Ethyl  alcohol,  production  by  fermentation, 
10,  11,  21,  302,  396,  461 
,  production  in  the  intestine, 

400 
,  passage  of,  into  milk,  539 
,  behavior    in     the     animal 

body,  632,  754 
,  action  on  the  secretion  of 
gastT'ic   juice,    351,    352, 
365,  369 
,  action    on    the    pancreatic 

juice,  385 
,  action  on  the  muscles,  465, 
466 


GENERAL   INDEX. 


789 


Ethyl  alcohol,  action  on  metabolism,  754 
,  action  on  digestion,  369 
,  action  on  proteins,  40,  41 
Ethyl   benzene,   behavior  in  the  animal 

body,  634 
Ethylene  glycol,  relation  to  formation  of 

glycogen,  291 
Ethylenimine.    See  Spermine. 
Ethylidene-lactic    acid,    460.      See    also 

other  lactic  acids. 
Ethyl  mercaptan,  behavior  in  the  animal 

body,  632 
Ethyl-sulphuric    acid,    behavior    in    the 

animal  body,  631 
Ethyl  sulphide,  formation    from   protein, 
28,  30,  33 
,  behavior    in    the   animal 
body,  612 
Euglobulin,  179,  ISO 
Euxanthic  acid,  122,  638 
Euxanthon,  638 

Euxanthon  glucuronic  acids,  610 
Excrementsr408,  410,  717,  718 

in  dogs  with  biliary  fistula,  407 
Excreta,  of  the  animal  organism,  715-720 
,  division  by  the  various  channels, 
717 
Excretin,  410 
Excretolic  acid,  410 
Exostosis,  438 
Expectorations,  713,  714 
Extinction  coefficient,  218 
Extracellular  action  of  enzymes,  22 
Exudates,  256-265 
Eye,  489-494 

Fseces.    See  Excrements. 

Fat,  origin  in  the  body,  442-446 

,  general  properties,  detection  and  oc- 
currence, 131-138 

,  relation  to  work,  471-474 

to  the  formation  of  glycogen, 
291,  292 

,  calorific  value  of,  723-728 

,  nutritive  value  of,  723-728,  747,  748, 
751,  752 

,  rancidity  of,  133, 

,  absorption  of,  421-427 

,  behavior  with  gastric  juice,  363,  364 

,  behavior  with  pancreatic  juice,  388- 
390 

,  saponification  of,  133,  389,  423 

,  action  of.  on  the  secretion  of  bile,  308 

,  action  of,  on  the  secretion  of  gastric 
juice,  349-351 

,  action  of,  on  the  secretion  of  pan- 
creatic juice,  384,  385 

,  iodized,  behavior  of,  in  the  animal 
body,  442,  538 

,  estimation    of,    136,    137,    526,    527 

,  metabolism  of,  in  activity  and  at  rest, 
471 

,  metabolism  of,  in  starvation,  729 

,  metabolism  of,   with  various   foods, 
740,  741,  770,  771 


Fat,  sugar  formation  from,  304-306 
Fat  formation,  from  proteins,  442-446 

,  from  carbohydrates,  442- 
446 
Fat-sweat,  692 

Fatty  acids,  general  properties,  detection 
and  occurrence,  131-138, 
441 
,  solubility  in  bile,  422,  423- 
,  absorption  of,  422 
,  sjTithesis,  444,  445 

to  neutral  fats,  421 
Fatty  deseneration,  443 
Fatty  infiltration,  282,  283,  443 
Fatty  series,  behavior  of  members  in  the 

animal  bodv,  629 
Fatty  tissue,  441,  446 

,  behavior  with  gastric  juice, 
360 
Feathers,  73 

,  pigments  of,  692 
Fehling's  solution,  116,  660 
Fellic  acid,  318 

Fermentation,  10,  11,  20,21, 109,  111,  11.5 
in  the  intestine,  397,  399 

403,  404 
in  the  urine,  677,  678 
in  the  gastric  contents,  371, 

373 
.    See  also  various  fermen- 
tations, alcoholic,  etc. 
Fermentation  lactic  acid,  properties,  occur- 
rence, etc.,  460- 

462 
in     the    gastric 
contents,  353 
in  the    souring 
of  milk,  460, 
515, 516 
,  detection  in  the 
gastric     con- 
tents, 374,375 
Fermentation  saccharometer.  664 
Fermentation  saccharomanometer,  664 
Fermentation  test  in  the  urine,  657,  663 
Ferments  in  general,  9-23 
inorganic,  14 
.    See  various  enzymes. 
Ferratin,  282 
Ferrine,  282 

Fevers,  elimination  of  ammonia  in,  625 
,  elimination  of  uric  acid  in,  569 
,  elimination  of  urea  in.  54S 
,  elimination  of  potassium  salts  in, 

623 
,  metabolism  of  proteins  in,  543 
Fibre,  crude,  utilization  of,  427 
Fibres,  elastic,  in  sputum,  714 

,  reticulate,  428 
Fibrin,  36,  171.  174.  183,  225,  228,  230 
,  occurrence  in  transudates,  260 
,  Henle's.  495 
Fibrin  coagulation,  175-178,  225-235 
Fibrin  calculi,  411,  682 
Fibrin  digestion,  356,  373,  392-394 


790 


GENERAL  INDEX. 


Fibrin  ferment,  16. 175, 176, 177, 183, 228- 

234 
Fibrin  formation,  175-178,  225-235 
Fibrin  globulin,  173,  175,  177,  183 
Fibrm  soluble.     See  Serglobulin. 
Fibrinogen,  36,  172-177, 183,  228-230,  250 
Fibrinolysis,  173,  175 
Fibrinoplastic  substance.   See  Serglobulin. 
Fibroin,  37,  81,  82 
Fischer-Weidel's  reaction,  160 
Fish-bones,  'f38 
Fish-eggs,  37,  504,  509 
Fish-scales,  81,  160 
Fish,  bile  of,  310,  328 

,  spermatozoa  of,  63,  151,  497 
,  swimming-bladder  of,  160,  710 
,  visual  purple  of,  490 
Fish-glue,  77 
Flesh,  metabolism,  in  starvation,  729 

,  metabolism,    with    various    foods, 
739-753 
Flesh  quotient,  476 
Florence's  sperma  reaction,  49.6 
Fluoride  plasma,  231 
Fluorine  in  bones,  436 

in  enamel,  440 
Fly-maggots,  formation  of  fat  in,  443 
Foods,  influence  on  the  secretion  of  intes- 
tinal juice,  377 
,  influence  on  the  secretion  of  bUe, 

308 
,  influence  on  the  secretion  of  gastric 

juice,  350,  351 
,  influence  on  the  secretion  of  pan- 
creatic juice,  382,  383 
,  influence  on  the  secretion  of  mUk, 

536 
,  influence  on  the  elimination  of  vu-ic 

acid,  569 
,  influence    on    the   elimination   of 

urea,  548 
,  influence   on   the   elimination   of 

purine  bases,  578 
,  influence  on  fseces,  408,  409,  417, 

418,421,719, 
,  influence  on  metabolism,  762 
,  requirements,  763-771 
,  various,  739-753 
,  insufficient  supply  of,  734-739 
Foodstuffs,  necessity  of,  715 

combustion  heat  of,  723-728 
Formaldehyde,  formation  in  plants,  1,  114 
,  action  upon  proteins,  50 
,  combination  with  urea,556 
,  relation   to   sugar   forma- 
tion, 113,  114 
Formic  acid  in  gastric  contents,  376 

,  passage  of,  into  urine,  607, 
629 
Frog's  eggs,  509 

membrane  of,  66 
Fructose.     See  Levulose. 
Fruit  sugar.     See  Levulose. 
Fundus  glands,  349,  363,  364 
Fungi,  glycogen  in,  288 


Fungi,  tyrosinase  in,  18 
Fumaric  acid,  32; 
Furfuracrylic  acid,  637 
Furfuracryluric  acid,  637 
Fiu"furol,  from  pentoses.  111 

,  relation    to   Pettenkofer's  bile- 
acid  tests,  312 

,  reagent  for  urea,  555 

,  behavior  in  the  animal  body,  637 
Fuscin,  491 

Galactonic  acid,  120 
Galactose,  106,  120,  524 

,  from  cerebrins,  484,  485 
,  relation  to  formation  of  glyco- 
gen, 293 
,  passage  of,  in  the  urine,  665 
Galactosamine,  71,  121 
Galactosides,  108 
Gallacetophenon,  behavior  in  the  animal 

body,  038 
Gallic  acid,  behavior  in  the  animal  bodv, 

597,  637 
Gallois's  inosite  test,  459 
Galtose,  108 

Gas,  exchange  of,  in  various  ages,  755-757 
,  exchange  of,  through  the  skin,  695 
,  exchange  of,  in  starvation,  730,  731 
,  exchange  of,  in  various  conditions  of 
the  body,  445,  446,  472,  720,  721, 
730,  733,  757,  759,  761 
,  exchange  of,  in  the  muscles,  468,  472 
,  exchange  of,  starvation  requirement 
of,  733 
Gases  of  the  blood,  696-702 

of  the  intestine,  403,  404 
of  the  bile,  329,  702 
of  the  urine,  626,  702,  703,  712 
of  the  hen's  egg,  509,  510 
of  the  lymph,  251,  702 
of  the  muscles,  465,  468 
of  the  transudates,  259,  712 
of  the  stomach,  372 
Gastric  contents.     See  Chyme. 
Gastric  fistula,  350,  352 
Gastric  juice,  349 

,  composition  of,  353,  354 

,  secretion  of,  349-352 

,  estimation  of  acidity  of,  373, 

374,  376 
,  relation  to  intestinal  putre- 
faction, 371,  406-408 
,  action  of,  356-363,  366-371 
Gastric  lipase,  363 
Gastric  mucosa,  348 
Gelatine,  28,  77-80,  100,  582 

,  relation  to  glycogen  formation 

291  * 

,  relation  to  coagulation  of  blood, 

233 
,  putrefaction  of,  401 
,  nutritive  value  of,  745 
,  behavior  with  gastric  juice,  360 
,  behavior  with  pancreatic  juice, 
391,  395 


GENERAL  INDEX. 


791 


Gelatina  in  the  egg,  511 
Gelatine  and  the  (leteetion  of  trypsin,  290 
Gelatine-forming  substances.     See  Colla- 
gen. 
Gelatine  peptones,  57,  58,  79 
Gelatine  sugar.    See  GlycocoU. 
Gelatinous  tissues,  429 
Gelatoses,  79 

,  relation  to  blood  coagulation, 
171 
Generation,  organs  of,  495-513 
Gentisic  acid,  599 

,  behavior    in    the    animal 
body,  637 
Gentisic  aldehyde,  599 
Gerhardt's  diacetic  acid  reaction,  672 
Globan,  46 

Globin,  62,  99,  197,  208 
Globulins,  36 

,  general  characteristics,  45 
,  in  starvation,  187 
,  in  urine,  643 
,  in  protoplasm,  140 
.    See  also  the  different  globu- 
lins. 
Globuloses,  52 
Glucalanine,  81 
Glucase,  21,  185,  345,  346 
Glucocyanhydrin,  106 
Glucoheptose,  106 
Gluconic,  acid,  105,  114,  300 
Glucosamine,  108,  120,  121 

from  chit  in,  120 

from  proteins,    32,    65,    67, 

500,  506-508 
in  diabetes,  300 
relation    to    formation    of 
glycogen,  294 
Gluconucleoproteids,  71,  72 
Glucoproteids,  36,  65-72,   141,  428,  430, 
500,  506 
,  relation    to    formation    of 
glycogen,  292,  293,  295 
Glucoproteose,  55 
Glucosaminic  acid,  121 
Glucosan,  115 

Glucose,  105,   106,   114.     See  also  Dex- 
trose. 
Glucosides,  13,  108,  110 
Glucosoxirae,  106 
Glucorone,  122 
Glucothionic  acid,  222,  272,  285,  431,  514, 

542 
Glucuronic  acid,  121,  122 

,  relation  to  formation  of 

glycogen,  291 
,  conjugated,      122,     609, 
632,  638,  666 
in  diabetes,  300 
in  blood,  184 
in  bile,  310,  325 
in  urine,  609,  632,  638, 
667 
Glutamic  acid,  88 
Gluteines,  77 


Gluten  casein,  100 
Gluten  proteins,  100 
Glutin  peptones,  58,  79 
Glutokyrin,  58 
Glutose,  108 
Glutinase,  391,  392 
Glutinic  acid,  78 
Glyceric  acid,  630 
Glycerine  aldehyde,  80 
Glycerine  relation  to  formation  of  glyco- 
gen, 291 
relation  to  formation  of  sugar, 
305 
Glycerophosphoric  acid,  144,  247, 272,  277, 

325 
Glycerophosphoric  acid  in  urine,  608,  614 
Glyceroses,  114 
Glycine.    See  GlycocoU. 
Glycocholeic  acid,  312 
Glycochohc  acid,  310,  312 

,  occurrence      in      excre- 
ments, 404 
,  occurrence  in  bile  from 

various  animals,  328 
,  absorption  of,  426 
,  behavior    to    intestinal 
pvitrefaction,  406 
Glycocholates  from  rodents,  313 
GlycocoU,  83 

GlycocoU,  relation   to   formation  of  uric 
acid,  56S,  572 
,  relation  to  formation  of  urea, 

550,  551,  630 
,  synthesis  with,  3,  585,  586,  635 
Glycogen,  140,  287-307 

,  origin  of,  289-307 

,  relation  to  muscular  activity, 

468,  472 
,  relation   to   muscle   rigor,    466 
,  relation  to  pepsin  secretion,  365 
,  occurrence  in  sputmn,  714 
,  occurrence  in  leucocytes,  221 
,  occurrence  in  the  lungs,  713 
,  occurrence  in  the  lymph,  251 
,  occurrence  in  protoplasm,  140. 
148,  221,  268 
Glycol  aldehyde,  2 
Glycolysis,  185,  302,  303,  396 
Glycolytic  enz^^ne,  20,  185,  302,  303 
Glycosuria,  29'7-307 

,  alimentary,  298,  420 
Glycosuric  acid,  597 

Glycuronic  acid,  121.    See  Glucuronic  acid. 
Glvcvkilanine,  35,  395 
Glycylglycine,  35,  396,  630 
GlycyI-1-leucine.  35 
Glycyl-1-tyrosine,  35 
Glyoxji  diureide.    See  Allantoin. 
Glyoxylic  acid,  as  reagent,  42 
Gmelin's  test  for  bile-pigments,  321 

test  for  bile-pigments  in  urine, 
652 
Goat's  milk,  528,  529 
Gold  equivalent  of  the  proteins,  41 
Goose-fat,  absorption  of,  423 


792 


GENERAL   INDEX. 


Gorgonin,  82 

Gout,  elimination  of  uric  acid  in,  570 
Graafian  follicles,  498 
Grape-moles,  512 
Grape-sugar.     See  Dextrose. 
Guaiaconic  acid  ozonide,  649 
Guaiacum  blood  test,  648 
Guanase,  16,  271,  273,  571 
Guanidine,  32,  33,  501,  550 
Guanine,  158,  160 

in  urine,  578 
Guanine  gout,  160 
Guano,  160,  569 
Guano-bile  acids,  313 
Guanovulit,  510 

Guanylic  acid,  151,  153,  155,  156,  381 
Gulonic  acid  lactone,  121 
Gulose,  113,  118 
Gums,  various,  129 
•  ,  animal,  67 
,  animal,  in  urine,  608 
Gunning-Lieben's   acetone   reaction,    670 
Giinzberg's  reagent  for  free  HCl,  374 

Hsemagglutination,  195 
Hsemase,  20 
Hsemataerometer,  706 
Hsematin,  197,  209 

,  relation  to  bilirubin,  332 
,  relation  to  urobilin,  603 
,  neutral  hsematin,  208 
Hsematinogen,  216 
Hsematino meter,  217 
Hsematinic  acids,  210,  211,  325 
Hi3ematinic  acid  imide,  211,  320 
Hsematinic  acid  ester,  211 
Hsematocrit,  236 
Haematogen,  503,  510 
Haematoglobulin.    See  Oxyhseriioglobin. 
Haematoidin,  216 

,  relation    to    bilirubin,    216, 

320,  331 
,  occurrence  in  sputum,  714 
,  occurrence  in  corpora  lutea, 

498 
,  occurrence    in    excrements, 

409 
,  occurrence  in  sediments,  680 
Haematoporphyrin,  213 

,  relation  to  chlorophyl, 

197,  214 
,  relation    to    bilirubin, 

215,  320,  332 
,  relation    to    urobilin, 

214,  332,  603 
,  occurrence    in    urine, 

601,  649,  650 
,  occurrence    in    lower 
animals,  690 
Hsematoscope,  219 
Hsematuria,  648 
Hsemerythrin,  219 
H^min,  211,  649 
Haemin  crystals,  211,  649 
Haemochrom,  196,  199 


Hsemochromogen,  197,  208,  209 

,  occurrence  in  muscles, 
451 
Hsemocvanin,  219 
Haemoglobin,  36,  65,  197 

,  composition  of,  198 

,  properties  and  behavior,  202 

,  quantity  in  blood,  197,  242- 

247 
,  quantitative         estimation, 

217-219 
.    See    also    Oxyhaimoglobin 
and  the  combinations  of 
haemoglobin    with    other 
gases. 
Haemoglobinuria,  648 
Haemolysis,  193,  337 
Hajmolysins,  186,  193 
Hsemometer,  219 
Haemopyrrol,  197,  214 
Haemorrhodin,  207 
Hsemoverdin,  208 
Haser's  coefficient, 
Hair,  73,  685 

,  pigments  of,  689 
Hair-balls,  411 
Half-rotation,    109 
Hammarsten's  reaction  for  bile-pigments, 

322,  653 
Haptogen-membrane,  517 
Heat,  action  of,  on  metabolism,  761 

of  combustion  of  various  foodstuffs, 
723-727 
Helicoproteid,  71 
Heller's  albumin  test,  41 

albumin  test  applied  to  urine,  640 
Heller-Teichmann's  blood  test,  649 
Hemicelluloses,  130      , 
Hemicollin,  79 
Hemielastin,  76 
Hemiindigotin,  594 
Heminucleic  acid,  154 
Hemipeptone,  51 
Hemp-seed  calculi,  681 
Hen,  development  of  the  embryo  of,  511, 

512 
Hen's  egg,  502-512 

,  incubation  of,  510 
white  of  the,  506-510 
yoke  of  the,  502,  506 
Heptoses,  105 

Herring,  spermatozoa  of,  63,  498 
Heterolysis,  23 

Heteroproteoses,  51,  55,  56,  57 
Heterosyntonose,  100 
Heteroxanthine,  157 

in  urine,  579 
Hexone  bases,  63,  96-100 
Hexoses,  113-120 

See  also  the  various  hexoses. 
High  altitude,  action  on  the  blood,   245 
Hippokoprosterin,  337 
Hippomelanin,  688 
Hippuric  acid,  585 

,properties  and  reactions^  587 


GENERAL  INDEX. 


793 


Hipp  uric  acid,  estimation  of,  578 

,  formation  in  the  body,  3, 

585,  635 
,  cleavage  of,  4,  584, 587,  588 
,  occurrence  of,  585 
as  sediment,  679,  680 
Hirudin,  231,  233 
HLstidine,  63,  99,  100,  108 
Histidylhistidine,  35 
Histone,  27,  36,  61,  100,  208,  229,  270 

in  urine,  647 
Histozyme,  588 
Hofmann's  tyrosine  test,  91 
Hog-fat,  423 

,  absorption  of 
Hog-flesh,  475 
Holothuria,  mucin  of,  69 
Holozyme,  232 
Homocerebrin,  482^84 
Homogentistic  acid,  68,  90,  597-599 
Hoppe-Seyler's  CO  blood  test,  206 

xanthine  test,  160 
Horn,  73,  685 

Horn  substance.      See  Keratins. 
Horse's  milk,  529 
Human  milk,  529-535 

,  behavior    in    the    stomach, 
530,  531 
Humin  substances  in  urine,  600 
Himior,  aqueous,  264 
,  vitreous,  491 
Huppert's  reaction  for  bile-pigments,  322 
reaction    for   bile-pigments   in 
urine,  653 
Hyalines,  68 

of  the  walls  of  hydatid  cysts,  687 
of  Rovida's  substance,  141,  221, 
267 
Hyalogens,  65,  68 
Hyalomucoid,  490 
Hydatid  cysts,  687 
Hydrtemia,  246 
Hydramnion,  513 
Hydrazones,  107 
Hydrobilirubin,  320 

,  relation  to  urobilin,  603 
Hydrocele  fluids,  259,  264 
Hydrocephalus  fluid,  264 
Hydroquinone,  591.  639 
Hydroquinone   sulphuric   acid,   588,    592 
Hydrochloric  acid,  secretion  in  stomach, 
353,  364,  365, 373 
,  anti-fermentive  action 

of,  371 
,  action  of,  on  secretion 
of  pancreatic  juice, 
384 
,  action  of,  on  secretion 

of  bile,  308,  309 
, 'action  of,  on  pylorus, 

367 
,  material  of,  371 
,  quantitative      estima- 
tion in  gastric  con- 
tents, 375,  376 


Hydrochloric  acid,  reagents  for  free  HCl  in 
gastric  contents,  374 
Hydrocinnamic  acid,  behavior  in  the  ani- 
mal body,  585 
Hydrocyanic  acid,  action  on  peptic  diges- 
tion, 359 
,  action   on    tryptic   di- 
gestion, 393 
Hydrogen  in  putrefactive  and  fermentive 

processes,  5,  401,  403 
Hydrogen    peroxide,    decomposition    of, 
•    by  catalases,  7,  20 
Hydrogenases,  19 
Hydrolytic  cleavages,  9,  16 

.  See  also  the  various 
cleavages. 
Hydronephrosis  fluid,  542 
Hydroparacoumaric  acid,  597 

,  in        intestinal 
putrefaction, 
401 
Hydroxylamine,  poisoning  with,  584 
Hyoglycocholic  acid,  313 
HjTiergluciemia,  298,  299 
Hyperisotonic  solutions,  193 
Hypisotonic  solutions,  193 
Hypnotics,  relation  to  formation  of  gly- 
cogen, 291 
Hypogaeic  acid,  138 
Hyjjosulphites  in  the  urine,  612 
Hyposulphurous  acid  in  urine,  612,  631 
Hypoxanthine,  158 

,  properties,  161,  162 

,  passage  of,  into  urine,  578 

Ichthidin,  504,  509 
Ichthin,  509 
Ichthulin.  71,  504,  509 
Ichthylepidin,  81 
Ignotin,  457 
Icterus,  307,  333,  652 

,  urine  in. 
Immunity,  25,  186 
Incubation  of  the  egg,  510 
Indican  test,  Jaffe's,  594 

,  Obermeyer's,  594 
Indican,  urine,  592-594 

,  elimination   in   starvation,    404, 

592,  593 
,  elimination  in  disease,  592,  593 
Indigo,  402,  592,  .594 
,  in  sweat,  695 
,  in  urinary  sediments,  680 
Indigo  blue.     See  Indigo. 
Indigo  red,  594 
Indigo  sulphonic  acid,  595 
Indol,  properties,  402 

,  formation  from  protein,  30,  34,  102 
,  formation  in  putrefaction,  401,  402, 

588,  592 
,  formation  from  melanins,  689 
,  in  the  blood,  186 
Indolacetic  acid,  596 
Indolaminopropionic  acid,  30  102 
Indophenol  reaction,  18 


794 


GENERAL   INDEX. 


Indoxj'^1.     See  Indol. 
Indoxyl-glucuronic  acid,  592,  595 
Indoxyl  red,  594 
Indoxyl-sulphuric  acid,  592,  595 
Inosinic  acid,  155,  454,  457 
Inosite,  properties   and   occurrence,    458, 
459 
in  urine,  668 
,  relation  to  formation  of  glycogen, 
291 
Integral  factor,  574 
Intestinal  calculi,  410-412 
Intestinal  fistula,  377,  380,  399 
Intestinal  gases,  403,  404 
Intestinal  juice,  377-379 
Intestinal  mucosa,  377 
Intestine,  putrefactive  processes  in,  399- 
408,  586-590 
,  reaction  in,   399,   400,   406-408 
,  absorption  in,  406,  411-427 
,  digestive  processes  in,  396-405 
Intestine  nucleic  acid,  152 
Intracellular  enzymes,  22.     See  also  the 

various  organs. 
Inulin,  127 

,  relation  to  formation  of  glycogen, 

290 
,  relation  to  the  secretion  of  pepsin, 
365 
Inversion,  123,  361,  397,  419 
Invertases,   16,  185,  361,  378,  419 
Invert-sugar,  123 

Iodides  and  secretion  of  gastric  juice,  364 
Iodine  equivalent,  137 
Iodine,  passage  of,  into  milk,  539 
,  passage  of,  into  sweat,   695 
.  ,  passage  of,  into  saliva,  347 
,  action  upon  protein,  30 
,  in  the  blood, 187, 243 
,  in  glands,  27,  271,  275,  276 
Iodized  proteins,  30,  81,  275,  598 
Iodized  fats,  442,  538 
Iodoform,  behavior  in  the  animal  body, 
631 
test.  Gunning's,  670 
test,  Lieben's,  670 
lodogorgonic  acid,  82 
lodohgematin,  213 
lodospongin,  81 
lodothyreoglobulin,  275 
lodothyrin,  275,  276 

Ion  action,  15,  39,  168,  169,  359,  393,  464 
Iron  in  blood,  238,  239 
in  urine,  626 
in  new-born,  286,  535 
,  elimination  of,  325,  332,  347,  626 
and  blood  formation,  244,  245,  503 
and  bile  formation,  332 
,  absorption  of,  244,   245 

See  also  various  tissues  and  fluids 
Iron  starvation,  738 
Isobilianic  acid,  315 
Isocholanic  acid,  317 
Isocasein,  520 
Isocholesterin,  335,  337,  691,  692 


Isocreatinine,  454 

Isocysteine,  93 

Isodynamic  law,  726 

Isoglucosamine,  108 

Isoiactose,  16 

Isoleucine,  87 

Isomaltose,  125,  184,  344,  388 

in  urine,  608 
Tsosaccharin,    relation    to    formation    of 

glycogen,  291 
Isoaerine,  96 
Isotonic  solutions,  193 
Isotropovis  substance,  447 
Ivory,  440 

Jaffe's  indican  test,  594 

creatinine  test,  566 
Janthinin,  691 
Japanese,  nutrition  of,  764 
Jaune  indien,  122 
Jecorin,  147,  273,  283 

,  in  blood,  184 
Jequirity  bean,  25 
Jolles's  reaction  for  bile-pigments,  653 

Kathsemoglobin,  208 
Kephir,  524,  528 

,  anti-putrefactive  action,  405 
Kephir  lactase,  524,  610 
Kerasin,  482,  283,  484,  488 
Keratose,  73 
Keratins,  37,  73-75_,  685 

,  behavior  in  the  stomach,  360 
,  behavior  with  pancreatic  juice, 
396 
Ketones,  behavior  in  the  animal  body,  632, 

637,  638 
Ketoses,  105,  113,  119 
Kidneys,  541 

,  relation  to  formation  of  urea,  553 
,  relation  to  formation  of  hippuric 
acid,  586,  587 
Kinases,  16,176,231,232,233,379,383,386 
Kjeldahl's  method  of  determining  nitro- 
gen, 556 
Knapp's  titration  method,  662,  663 
Knee-joint  cartilage,  364-434 
Rnop-Hiifner's    method   for   determining 

urea,  562 
Koprosterin,  335,  337,  408 
Krinosin,  486 
Kumyss,  524,  528 
Kyestein,  680 
Kynurenic  acid,  597,  600 
Kyrins,  58,  59 
Kyroprotic  acids,  31 

Laborer,  diet  of,  763-770 
Laccase,  18 
Lactacidase,  21  * 

Lactalbumin,  36,  522,  523,  526 
Ijactase,  524 

in  the  intestine,  379,  419 

in  the  pancreas,  386 
Lactates.     See  I^actic  acids,  also  461-463 


GENERAN   INDEX. 


795 


Lactic-acid  fermentation,  110,  116,  371, 

373,  389,  400, 

460,  516,  524 

in  intestine,  397 

399 
in  stomach,  371, 

373 
inniilk,515,516, 
523,  524 
Lactic  acids,  460 

,  in  intestine,  397,  400 
,  in  urine,  460,  572,  608 
,  in  bones,   439 
,  in  stomach,  353,  374 
,  relation  to  formation  of  uric 
,      acid,  572,  573 
.    See  also  Paralactic  and  Fer- 
mentation lactic  acids. 
Lacto-caramel,  524 
Lacto-globulin,  522 
Lactolase,  21 

Lactone  of  saccharic  acid,  121 
Lactones  of  varieties  of  sugars,  105,  106 
Lactophosphocarnic  acid,  523 
Lactoprotein,  523 
Lactose.     See  Milk-sugar. 
Laiose,  665 

Lakey  color  of  blood,  225 
Lamb,  intestinal  fluid  in  the,  377 
Lanoceric  acid,  692 
Lanocerin,  692 
Lanolin,  337,  642 
Lanopalmitic  acid,  692 
Lanugo  hair,  512 
Lard,  absorption  of,  424 
Large  intestine,  extirpation  of,  426,  427 

,  secretion  of,  380 
Latebra,  502 

Laurie  acid,  131,  138,  51S 
Lead  in  the  blood,  239 
in  the  hver,  286 
passage  of,  into  milk,  539 
Lecithalbumins,  47,  349,  541 

,  relation   to   secretion   of 

gastric  juice,  349 
,  relation   to   secretion   of 
urine,  541 
Lecithans,  488 
Lecithins,  143,  144 

,  in  egg-yolk,  502,  503 
,  in  the  brain,  480,  488 
,  in  the  muscles,  463 
,  in  milk,  518,  532 
,  in  the  liver,  283 
,  importance  for  cells,  143,  193 
,  putrefaction  of,  146,  177,  404 
Legal's  acetone  reaction,  671 
Lens,  (see  Crystalline  lens),  492 
,  capsule  of,  69,  492 
,  fibres  of,  492 
Leo's  sugar,  665 
Lepidoporphyrin,  690 
Lepidotic  acid,  690 
Lethal,  138 


Leucaemia,  blood,  158,  247 

,  uric  acid,  elimination  in,  274, 

569,  570 
,  purine     bases    in,    158,    247, 
578 
Leucin,  85-87 

,  relation  to  formation  of  uric  acid, 

572 
,  relation  to  formation  of  urea,  550, 

551,  630 
,  passage  of,  into  urine,  614,  675 
,  behavior  in  the  animal  body,  550, 
551,  630 
Leucin  ester,  86,  87 
Leucin  ethylester,  87 
Leucinic  acids,  86 
Leucinimide,  87 
Leucocytes,  relation  to  absorption,  416 

,  relation  to  formation  of  uric 

acid,  570 
,  in  thymus  gland,  272 
Leucomaines,  24 

,  in  urine,  615 
,  in  muscles,  457 
Leuconuclein,  229,  270 
Leucylalanyl-glycine,  35 
Leucyl-1-tyrosine,  35 
Levolactic  acid,  460 
Levuline,  127 

Levulinic  acid,  67,  113,  152,  524 
Levulose,  106,  108,  118,  119 
,  in  urine,  664 
,  in  blood,  184 
,  relation  to  glycogen  formation, 

293 
,  absorption  of,  419,  420 
,  behavior  in  diabetics,  300 
,  in  transudates,  260,  512 
Lichenin,  127 

Lieben's  acetone  reaction,  670 
Lieberkiilm's,  alkali-albuminate,  48 

,  glands,  377 
Liebermann's  reaction  for  proteins,  43 
liiebermann-Burchard's  reaction  for  cho- 

lesterin,  336 
Liebig's  titration  method  for  estimating 

urea,  557-560 
Lienases,  273 

Ligamentum  nuchce,  75,  76,  429 
Lignin,  129 
Linoleic  acid;  131,  135 
Linolic  acid,  504 

Linseed-oil,  feeding  with,  442,  538 
Lion's  urine,  567 
Lipanin,  absorption  of,  423 
Lipase,  16 

in  blood,  185 
in  stomach,  363 
in  the  intestine,  379,  380 
in  the  liver,  284 
in  pancreatic  juice,  384,  388 
in  milk,  523 
Lipiawsky's     acetoacetic    acid    reaction, 
672 


796 


GENERAL   INDEX. 


Lipochromes,  186,  505 
Lipoids,  193 
Lipuria,  675 

Lithium,  in  blood,  166,  239 
Lithium  lactate,  463 
Lithium  urate,  575 
Lithobilic  acid,  411 
Lithofellic  acid,  318,  411 
Lithuric  acid,  616 
Liver,  280-287 

,  relation  to   coagulation  of  blood, 

172,  234,  235 
,  relation  to  formation  of  uric  acid, 

571,  572,  573 
,  relation  to  formation  of  urea,  550, 

551,  552,  554 
,  blood  of,  242,  296,  297 
,  proteids  of,  286,  287 
,  fat  of,  282,  283 

,  quantity  of  sugar  in,  295,  296,  297 
Liver  atrophy,  acute  yellow,  23,  284 

,  elimination  of  amino  acids 

in,  284,  285,  675 
,  elimination  of  ammonia  in, 

554 
,  elimination  of  urea  in,  554 
,  elimination  of  lactic  acid 

in,  461,  608,  609 
,  autolysis,  23,  284 
Liver  cirrhosis,  ascitic  fluid  in,  262,  263 
,  action  of,  on  the  elimina- 
tion   of    ammonia    and 
urea,  554 
Liver  extirpation,  elimination  of  ammonia 
with,  554,  572 
,  elimination  of  uric  acid 

with,  572,  573 
,  elimination  of  urea  with, 

554 
,  elimination     of     lactic 
acids  with,  460,  572, 
608 
,  action  on  formation  of 
bile,  330 
Lotahistone,  62 
Lung  catheter,  708 
Lungs,  703,  704,  713,  714 
Luteins,  505 

in  corjjora  lutea,  216,  498 
,  egg-yolk,  505 

in  blood-serum,  180 
,  relation  to  hsematoidin,  216,  505 
Lymph,  250-256 
Lymphagogues,  255 
Lymphatic  glands,  269 
Lymph-cells,  quantitative  composition  of, 
272 
.    See  also  Leucocytes. 
Lymph-fibrinogen.    See  Tisaue-fibrinogen. 
Lysalbinic  acid,  49 
Lysatine  and  lysatinine,  99 
Lvsinp.  33,  64.  98,  99,  186 
Lysines,  25,  186,  193 
Lysuric  acid,  98 
Lysyllysine,  35 


Mackerel,  flesh  of,  475 

,  sperm  of,  62,  63 
Madder,  feeding  with,  438 
Magnesium  in  urine,  625,  628 
in  bones,  436,  440 
m  muscles,  464,  475,  477 
.    See  also  various  tissues  and 
fluids. 
Magnesium  phosphate  in  intestinal  calculi, 
411 
in  urine,  619,  625, 

628 
in   urinary   calculi, 

680,  681,  684 
in     urinary     sedi- 
ments, 677,  679 
in  bones,  436,  440 
Magnesium  soaps  in  excrements,  408 
Malic  acid,  behavior  in  the  animal  body, 

544 
Maltase,  16,  125,  345,  346,  388 
Maltodextrin,  128 
Maltoglucase,  21,  185,  344,  346 
Maltose,  123,  124 

,  formation  from  starch,  124,  127, 

344,  388 
,  absorption  of,  419 
,  relation  to  glycogen  formation, 
293 
in  intestine,  397,  419 
,  occurrence  in  urine,  667 
Mammary  glands,  514,  537 
Mandelic  acid,  634 
Man  in  poorhouse,  diet  of,  770 
Mannite,  106 

,  relation  to  formation  of  glycogen, 
291 
Mannonic  acid,  114 
Mannose,  108,  113,  114,  118 
Mare's  milk,  530 

Margarine  and  margaric  acid,  134 
Marsh-gas,  formation  in  putrefaction,  30, 

401,  404 
Martamic  acid,  154.     See  also  Methane. 
Maschke's  creatinine  reaction,  565 
Meat  extracts,  action  on  secretion  of  gas- 
tric juice,  365 
,  constituents  of,  454,  456 
Meat,  utilization  in  intestinal  tract,  417 
,  calorific  value  of,  724-726 
,  digestibility  of,  368 
,  composition  of,  443,  444,  474-476 
.     See  also  muscles. 
Meconium,  410 
Medulla  oblongata,  486 
Melanins,  154,  688-690 
in  the  eye,  491 
in  the  urine,  651 
Melanogen  in  the  urine,  651 
Melanoidic  acid,  688 
Melanoidins,  27,  -30,  154 
Melanotic  sarcoma,  pigment  of,  688 
Melissyl  alcohol,  138 
Membranins,  69,  434,  492 
Menstrual  blood,  187,  242 


GENERAL  INDEX. 


'9'i 


Menthol,  behavior  in  the  anim:.!  body,  638 
Mercaptan,  from  proteins,  30,  33,  73,  401 
Mercapturic  acids.  63S 
Mercury  salts,  passage  of,  into   milk,   539 
,  passage  of,  into  sweat,  695 
,  action  on  ptyalin,  346 
,  action  on  trj-psin,  393 
Mesitylene,  behavior  in  the  animal  body, 

635 
Mesitylenic  acid,  635 
Mesitylenuric  acid.  635 
MesoporphjTin,  214 
Metaca.sein  reaction,  521 
Metabolism,  dependence  of  external  tem- 
perature upon,  761,  762 
in  various  ages,  755-758 
in  work  and   rest,   467-474, 

758-761 
in  different  sexes,  756 
in  starvation,  728-733     ' 
with  different  foodstuffs,  739- 

754 
in  sleep  and  waking,  761 
calculation  of  extent  of,  720, 
723 
Metalbumin,  499,  500 
Metallic  sols,  14 

Metaphosphoric  acid,  as  reagent  for  pro- 
teins, 41,  642 
Metazym,  232 
Methffimoglobin,  203,  218 

in  urine,  648 
Methal,  138 
Methane,  formation    in    putrefaction,  30 

401,  404 
Methose,  114 
Methylenitan,  114 

Methvlethvlmaleic    acid    anhvdride,    211 
Methylfurfurol.  336 
Methyl  glycocoU.     Ree  Sarcosin. 
Methylguanidine,  455,  457,  565 
Methylguanidin-acetic    acid.     See    Crea- 
tine. 
Methylhydantoic  acid,  631 
Methyl  iudol.     See  Skatol. 
Methylimidazol.  lOS 
Methyl   mercaptan    in  proteins,  30,   401, 

404 
Methyl  pentose.     See  Rhamnose. 
Methyl  p\Tidjne,  behavior  in  the  animal 

body.  634,  637 
Met hvl-i ) vridvl-amraoniiun    hydroxide, 

639 
Methylthiophene,  336 
Methyl ura mine,  455,  457,  565 
Methyl  xanthine.  157,  579, 
Micrococcus  restituens,  415 
Micrococcus  urea,  677 
Micro-organisms    in   intestinal    tract,  24, 

371,  398,  399,  405,  408 
Milk,  515-542 

,  secretion  of,  536,  537 

,  consumption   of,   in   intestine,  417, 

425.  530,  531 
,  blue,  540 


Milk,  anti-putrefactive  action  in  intestine, 
405,  589 
in  disease,  5.39 
,  passage  of  foreign  bodies  into,  538 
,  behavior  in  the  stomach,  367,  371, 

530,  531 
.    See  also  various  kinds  of  milk. 
Milk-fat,  517,  5.30 

,  formation  of,  537,  538 
Milk-globules  from  cow's  milk,    516,    517 

from  human  milk,  530 
Milk-plasma,  518 
Milk-sugar,  135,  524 

,  relation  to  formation  of  gly- 
cogen, 293 
,  properties  of,    524, 
,  fermentation  of,  524 
,  calorific  value  of,  724 
,  quantitative     estimation    of, 

527 
,  absorption  of,  419 
,  passage   of,   into   unne,    293, 

420,  524,  665 
,  origin  of,  538 
Millon's  reagent,  42,  43 
Mineral  acids,  alkali-removing  action   of, 
544,  624 
,  action   on   the   elimination 
of  ammonia,  544,  624 
Mineral  bodies,  elimination  in  starvation, 
620,  623,  731 
,  insufficient  supply  of,  734- 
738. 

See     also     the     various 
fluids,       tissues,       and 
j  uices. 
Modified  proteins,  40 
Molisch's  sugar  test,  117 
Monaniino  acids,  83-96 

behavior  in  animal  body, 
305,    462,    550,    572, 
614,  630 
Monosaccharides,  105-123 
Moore's  sugar  test,  115 
Morner-Sjoqvist's  method    of    estimating 
urea,  561 
method    of    estimating 
acidity,  376 
Momer's    tjTOsins    test,    89.      See    also 

Denige's. 
Morphine,  passage   of,    into    urine,    608, 
639 
,  passage  of,  into  milk,  539 
Mucic  acid,  120,  128,  524 

,  relation  to  formation  of  gly- 
cogen. 291 
Mucilages,  vegetable,  129 
Mucin,  36,  65-68 

in  sputum,  714 
in  cysts.  501 
in  urine,  615,  647 
in  .salivarj-  glands,  339,  340 
Mucin-like  substances  in    bile,    309,    310, 
329 
in  urine,  615,  646 


798 


GENERAL   INDEX. 


Mucin-like  substances  in  kidneys,  541,542 
Mucinogen,  66,  340,  509 
Mucinoids.    See  Mucoids. 
Mucin  peptone,  67,  360 
Mucoids,  36,  65,  68 

in  ascitic  fluids,  262 

in  the  vitreous  humor,  491 

in  the  cornea,  434 

in    connective    tissue,    428-430, 

435 
in  the  hen's  egg,  506,  508 
in  cysts,  498-501 
Mucoproteoses,  360 
Mucous  glands,  66,  339,  348,  380 
Mucous  membranes  of  the  stomach,  348 
Mucous  tissue,  430 
Mucus  of  the  bile,  310,  327 

of  the  urine,  542,  615,  647 
of  synovial  fluid,  265 
Mulberrj^  calculi,  681 
Murexide  test,  575 

Muscle,  coagulation     of.         See     Muscle- 
plasma. 
,  chemical  tones  of,  467 
,  permeability  of,  465 
Muscle-fibres,  447,  465 

,  permeability,  465 
Muscle-pigments,  453 
Muscle-plasma,  448,  449,  452,  453 

,  coagulation  of,  448,  452, 
453,  466,  477 
Muscle  rigor,  465 
Muscle-serum,  448 
Muscle-stroma,  451 
Muscle-sugar,  459 
Muscle-syntonin,  451 
Muscles,  Bowman's  disks,  448 
,  non-striated,  477 
,  striated,  447-477 
,  blood  of,  242,  468 
,  chemical  processes  in  work  and 

rest,  467-474,  759 
,  chemical  processes  in  rigor,  465 
,  proteins  of,  447-454,  466,  470 
,  extractives  of,  453-464 
,  enzymes  of,  453 
,  pigments  of,  453 
,  fat  of,  463.  472-474 
,  gases  of,  465,  468 
,  calorific  value  of,  725-727 
,  mineral  bodies  of,  464,  477 
,  amount  of  water  in,  476 
,  composition  of,  474-477 
Muscular  energy,  origin  of,  472-474 
Musculamine,  457 

Muscular  force,  chemical  processes  in  mus- 
cles, 467-474 
,  action  of,  on  urine,  544, 

564,  567,  608,  616 
,  action  of,  on  metabolism, 
470-474,  758-761 
Musculin,  450,  452,  478 
Mussels,  glycogen  of,  287 

,  muscles  of,  477 
Mutton-fat,  feeding  with,  442 


Mutton-fat.  absorption  of,  423,  424 

Myeline  forms.  481 

Myelines,  481,  488 

Myoalbumin,  449,  451 

Myogen,  453 

Myogen  fibrin,  449,  453,  466 

Myoglobulin,  449,  451 

Myohaematin,  454 

Myoproteid,  452,  453 

Myosin,  36,  221,  449,  450,  452,  466 

,  absorption  of,  413 
Myosin  ferment,  452,  453 
Myosin  fibrin,  449,  452 
Myosinogen,  452 
Myosinoses,  52 
Myricin,  138 
Myricyl  alcohol,  138 
Myristic  acid  in  animal  fat,  131 

in  butter,  518 

in  bile,  325 

in  wool-fat,  692 
Mytolin,  451 
Myxfjedema,  276 
Myxoid  cysts,  498 

Nails,  73,  685 

Naphthalene,  action  on  urine,  639 

,  behavior  in  the  animal  body, 
633 

Naphthalene   sulphocliloride   as   reagent, 
675 

Napthalene  sulpho  derivatives  of  amino 
acids,  92,  675 

Napthol-glucuronic  acid,  639 

Napthol,  reagent  for  sugar,  117,  659 

,  behavior   in    the   animal    body, 
609,  639 

Napthyl    isocyanate    compound    of    the 
amino-acids,  92 

Narcotics,  relation  to  glycogen  formation, 
291 

Native  proteins,  40 

Navel  cord,  mucin  of,  66,  67,  429 

Negative  phase,  234 

Neosine,  457 

Neossin,  69 

Neozym,  232 

Nerves,  479,  480,  488 

Neuridine,  481,  486.  502 

Neurine,  145,  277,  485,  615 

Neurochitin,  489 

Neuroglia,  480 

Neurokeratin,  73,  480,  488,  489 

Neutral  fats.    See  Fats. 

Nicotine    action   upon   the   gases   of   the 
stomach,  372 

Nitrates  in  the  urine,  623 

Nitric-oxide  htemoglobin,  207 

Nitriles,  behavior  in  the  animal  body,  631 

Nitro-benzaldehvde,  behavior  in  the  ani- 
mal  body,  636 

Nitro-benzene,  635 

Nitro-benzoic  acid,  636 

Nitro-benzyl  alcohol,  638 

Nitro-celluiose,  130 


GEXEPwlL  INDEX. 


799 


Nitro-hippiiric  acid,  636 
Nitro-phenol,  635 

Nitro-phenyl-propiolic  acid,  reagent      for 

sugar,  117, 

6.59 

,  behavior    in 

the  animal 

body,      592, 

595 

Nitro-toluene  sulpho-compounds  of  amino 

acids,  92 
Nitro-tolulene,    behavior   in    the    animal 

body,  638 
Nitrogen,  combined,  quantity  of,  in  intes- 
tinal   evacuations, 
717,  718 
,  in  meat,  443,  476 
,  in  protein  bodies,  27 
,  m  urine,  548,  549 
,  estimation      of,      in 
urine,     5.56-561 
Nitrogen  deficit,  718 

Nitrogen  elimination  in  work    and    rest, 

470-473,  7.58,  7.59 

in  starvation,  728- 

730 
with  various  foods, 

739-752 
through   the  intes- 
tine,    417,     418, 
717,  718 
through  the  urine, 
548,     549,      611, 
612,      620,      622, 
717-719 
through  the  epider- 
mis, 718 
through  the  sweat, 
694; 718 
,  relation  to  the  elim- 
ination of   phos- 
phoric  acid,   620 
,  relation  to  the  elim- 
ination    of     sul- 
phuric acid,  622, 
719 
,  relation  to  digestive 
activity,     624, 
717,  718,  762 
Nitrogen,  free,  in  blood,  696 

,  in  intestine,  403 
,  in  .stomach,  372 
,  in  secretions.  702 
,  in  transudates,  702 
,  in  urine,  626 
Nitrogen,  residual,  183 
Nitrogenous  equilibrium,  718.     See  also 

Chap.  XVIIl 
Nitroso-indol  nitrate,  402 
Non-striated  muscles,  477 
Non-biuret  giving  products,  54,  394,  417 
Norisosaccharic  acid,  121,  504 
Nova  in.  457 
Nubecula,  .542,  615 
Nucleases,  153,  271,  391 


Nucleic  acids,  71,  1.50-151,  1.56,  195 

in  the  urine,  647 
Xuclein  bases,  156-164 

in  blood,  1.58,  186 
in  the  m-ine,  578 
Nucleins,  72,  149 

,  relation  to  elimination  of  allox- 

uric  bases,  578 
,  relation    to    formation    of    uric 

acid.  570,  571 
,  relation  to  elimination  of  P.Or, 

619,  620 
,  behavior  with  gastric  juice,  72, 

149,  150,  3.50 
,  behavior  with  pancreatic  juice, 
394,  .395 
Nuclein  plates,  222 
Xucleoalbvunins,  .36,  46,  141,  1.50 

in  the  bile,  310,  329 
in  the  liver,  281 
in  the  urine,  646 
in  the  kidneys,  .541,  542 
in  protopla.sm,  141 
in  transudates,  2.58,  261 
,  behavior  in  pepsin  diges- 
tion, 46,  150,  522,  532 
Nucleoglucoproteids,  71,  72 
Nucleohistone,  62,  221,  269 

,  relation  to  coagulation  of 
blood,  229 
in  urine,  647 
Nucleoproteids,  36,  71,  72,  142,  149 
in  the  liver,  282 
in  gastric  juice,  354,  355 
in  blood.  172,  178 
in  bile,  329 

in  mammarj'  glands,  514 
in  muscles,  451,  477 
in  the  kidneys,  541 
in  the  pancreas,  149,  150, 

381 
in  protopla.sm,  142 
in  cell  nucleus,  142,  149 
in  thyroid  gland,  275 
in  thymus,  270 
,  behavior  in  pepsin  diges- 
tion, 71,  1.50 
,  behavior  with  pancreatic 
juice,  395 
Nucleotin,  154 

Nucleotin  phosphoric  acid,  154 
Nucleon,  457,  477 

in  milk,  523,  532 
Nucleosin,  165 
Nutrition  requirements,  763-771 

,  of  man,  7.39-7.53 
Nylander's  reagent.    See  Almen-Bottger's 
sugar  test. 

Obermeyer's  indican  test,  594 
Obermuller's  chole-sterin  reaction,  337 
Oblitin,  457 

Odoriferous  bodies  in  the  urine,  586 
CEdema,  subcutaneous,  fluid  from,  265 
Oertel's  diet  cure,  for  corpulency,  770,  771 


800 


GENERAL   INDEX. 


Oesophageal  fistula,  350 
Oleic  acid,  135 
Olein,  134 
Oleodistearin,  132 
01iga?mia,  246 
Oligocythseniia,  246 
Oliguria,  628 
Olive  oil,  absorption  of,  423 

,  action  on  the  secretion  of  bile, 
308,  309 
Onuphin,  69 
Oocyanin,  509 
Oorodein,  509 
Opalisin,  523,  531 

Opium,  passage  of,  into  the  milk,  539 
Optograms,  491 
Orcin  test,  111,  666 
Organic  acids,  behavior  in  theanimal  body, 

624,  629-631 
Organized  proteids,  742,  743 
Organs,  distribution  of  the  blood  in,  249 
loss  of  weight  in  starvation,  732 
Organs  of  generation,  495-513 
Ornithine,  97,  635 
Ornithuric  acid,  97,  635 
Orotic  acid,  523 
Orthonitrophenylpropiolic  acid.    See  Nit- 

rophenylpropiolic  acid. 
Orylic  acid,  523 
Osaminic  acid,  107 
Osamines  of  varieties  of  sugar,  107 
Osazones,  107 
Osmosis,  relation  to  absorption,  427 

,  relation    to    lymph    formation, 
255,  256 
Osmotic  pressure  of  blood,  189,  159 

of  urine,  546 
Osones,  107 
Ossein,  76,  435 
Osseoalbvmioid,  435 
Osseomucoid,  66,  435 
Osteomalacia,  438,  439 
Osteoporosis.     See  Osteosclerosis. 
Osteosclerosis,  438 
Otoliths,  494 
Ovalbumin,  32,  507 

,  relation  to  glycogen  formation, 
290 
Ovarian  cysts,  498-502 
Ovaries,  498 
Ovoglobulin,  33,  506 
Ovomucoid,  68,  508 
Ovomucin,  506 
Ovovitellin,  36,  503 
Ovum,  502-512 
Oxalate  calculi,  680,  681 
Oxalate  of   lim.e.     See   Calcium   oxalate. 
Oxalates,  action  on  blood  coagulation,  171, 

225 
Oxalic  acid,  in  the  urine,  582,583,678,680 
behavior  in  the  animal  body 
582,  783,  629 
Oxaluric  acid,  568,  582 
Oxaluric-acid  amide,  32 
Oxamide,  29,  32 


Oxaminic  acid,  32 
Oxidases,  8,  16-19,  185 

.     See  also  the  tissues  and  fluids. 
Oxidation  ferment.     See  Oxidases. 
Oxidations,  3-9,  17-19,  629-631,  633,  703 

in  diabetes,  300 
Oximes,  106 
Oxonic  acid,  568 

Oxvacids,  formation  in  putrefaction,  30, 
401 
,  detection  of,  597 
,  passage  of,  in  \n-ine,  401,  596 
,  in  the  sweat,  694 
Oxybenzoic  acid,  behavior  in  the  animal 

body,  635,  636 
Oxybenzenes,  633 
Oxbutyric  acid,  668,  072,  673 

,  detection  and  estimation, 
673-675 
in  the  blood,  702 
,  passage  of,  into  the  urine, 
625,  668,  669,  672,  673 
Oxydiaminosuberic  acid,  1.9,  33 
Oxydiaminosebacic  acid,  282 
Oxyethylsulphonic  acid,  behavior  m  the 

animal  body,  632 
Oxyfatty  acids  in  animal  fat,  131 
Oxygen,  consumption,  199,  704,  705 

in  work  and   rest, 

468,  472 
in  starvation,  729, 

730 
through    the    skin, 
695 
Oxygen,  activity  of,  3-8 

in  the  blood,  697,  704-709 

in  the  intestine,  403 

in  the  lymph,  251,  702     ' 

in  the  stomach,  372 

in     the     swimming-bladder     of 

fishes,  710 
in  secretions,  702,  703 
in  transudates,  703 
,  tension  of,  in  blood,  203,  698 
,  lack  of  action  on  protein  destruc- 
tion, 461,  569,  611 
,  lack  of,  action  on  elimination  of 

lactic  acid.  461,  469,  608 
,  lack  of,  action  on  elimination  of 
sugar,  461 
Oxygen  capacity,  specific,  711,  727 
Oxygen-carriers,  7,  648,  649 
Oxygen,  calorific  value  in  the  combustion 

of  different  foo  s,  623-624,  723 
Oxygen,  consumption  in  the  blood, 
Oxygenases,  17 
Oxyhfematin,  210 
Oxyhspmocyanin,  219 
Oxyhaemoglobin,  198 

dissociation  of,  198-200, 
704,  705 
,  properties  and  reactions, 

198-202 
,  quantity  of,  in  the  blood, 
196,  197,  239, 241-246 


GENERAL   INDEX. 


801 


Oxyhsemoglobin,  quantity  in  the  muscles, 

45 -i 
,  passage  of,  into  the  urine, 

64S 
,  behavior     with     gastric 

juice,  360 
,  behavior    with    trypsin, 

Oxj'^hydroparacoumaric  acid,  597 

Oxymandelic  acid,  597,  600 

Oxymonamino  acids,  29,  33,  95 

Oxymonaminosuberic  acid,  29,  96 

Oxymonaminosuccinic  acid,  29,  96 

Oxynaphthalene,  632 

Cxyphenyl-acetic  acid,  90,  401,596,597, 
637 

C  xjTjhenylaminopropionic  acid.  See  Ty- 
rosine. 

Oxyphenylethjdamine,  30,  54 

Oxyphenylpropionic  acid,  401,  597,  637 

Oxyproteic  acid  in  urine,  612,  613 

Oxj'^proteins,  31 

Oxyprotosulphonic  acid,  31 

Oxji^yrrolidincarboxyUc  acid,  34,  102 

OxyquinoHne,  638 

OxyquinoUnecarboxylic  acid,  600 

Ozone,  3 

Ozone  traasmitter,  201 

Palmitic  acid,  134 
Palmitin,  134 
Pancreas,  381 

,  relation  to  glycolysis,  185,  302, 

396 
,  extirpation  of,  action  on  absorp- 
tion, 
,  extirpation    of,    elimination    of 

sugar,  418,  421,  425 
,  pepsin,  385 

,  change  during  secretion,  381 
Pancreatic  calculi,  396 
Pancreatic  diabetes,  301,  302 
Pancreatic  diastase,  388 
Pancreatic  protein,  150,  381 
Pancreatic  rennin,  396 
Pancreatic  casein,  396 
Pancreatic  juice,  382-388 

,  secretion  of,  382-385 

,  enzymes  of,  386 

,  action  on  foodstuffs,  387- 

395 
,  action  upon  peptides,  35, 
396 
Parabamic  acid,  568 
Paracasein,  522 
Parachyrao.sin,  361,  362 
Paracresol,  formation  in  putrefaction,  401, 

588 
Paraglobulin.    See  Serglobulin. 
Paraglycocholic  acid,  313 
Parahaemoglobin,  201 
Parahistone,  63 
Paralactic  acid,  460 

,  relation  to   formation  of 
uric  acid,  572,  573 


Paralactic  acid,  properties  and  occurrence, 
460-463 
,  formation  from  glycogen, 

461,  466,  467 
,  formation  in  osteomalacia 

bones,  439 
,  formation  in  muscle  dur- 
ing work,  468-470,  472, 
473 
,  formation  in  rigor  mortis, 

467 
,  formation  in  lack  of  oxy- 
gen, 460,  461,  469 
,  formation  in  animals  with 
extirpated   livers,    460, 
461,  572 
,  passage  of,  into  the  urine, 
460-462,  572,  608 
Paralbumin,  275,  500 
Paralytic  saliva,  340 
Paraminophenol,  633 
Paramucin,  500 
Paramyosinogen,  450,  452 
Paranuclein.      See  Pseudonuclein. 
Paranuclsic  acid,  522 
Paraox^T^henylacetic  acid,  90,    401,    5C6, 

597,  637 
Paraoxvphenvlaminopropionic    acid,    89, 

401,  o96,  597,  637 
Paraoxj^aropiophenone,  behavior  in  ani- 
mal body,  638 
Parapeptone,  359 
Paraxanthine,  157,  580 

in  urine,  578,  580 
Parenterally  introduced  protein,  412 
Parietal  or  delomorphic  cells,  349,  364 
Parotid,  339 
Parotid  saliva,  341 
Parovarial  cysts,  501 
Partition  of  the  nitrogen  in  the  urine,  548, 

549,  568,  569,  611 
Peas,  utilization  in  the  intestine,  421 
Pemphigus  chronicus,  265 
Penicillium  glaucum,  86 
Pennacerin,  692 
Pentacrinin.  691 

Pentamethylendiamine.    See  Cadaverin. 
Pentosanes,  110 

digestion  of,  427 
Pentoses,  110 

,  relation  to  glycogen  formation, 
111,  290 
in  blood,  184 
in  urine,  110,  666 
in  pancreas,  110 
in  nucleic  acids,  152,  155 
in  nucleoproteids,  72,  110,  285, 
514 
Penzoldt,  acetone  reaction,  671 
Pepsin,  354-361 

,  detection  in  gastric  contents,  373 
,  quantitative  estimation,  357 
,  occurrence  in  the  urine,  426,  615 
Pepsin  charge,  in  the  stomach,  364 
,  in  the  pancreas,  385 


802 


GENERAL   LXDEX. 


Pepsin  cells,  349 

Pepsin  digestion,  356-362 

,  products  of,  52,  53,  359 
Pepsin  glands,  348 
Pepsiu-glutin  peptone,  57 
Pepsin-hydrochloric  acid,  360,  361 
Pepsin-like  enzyme,  354 
Pepsinogen,  364 
Pepsin  peptones,  51,  57 
Pepsin  test,  356 
Peptides,  35,  54,  394,  395,  416,  614 

,  relation  to  trypsin,  35,  395 
Peptochondrin,  433 
Peptones,  29,  30,  36,  49-61,  359 

,  assimilation  of,  413-417 
,  absorption  of,  414,  415 
,  passage  of  into  m'ine,  414,  643 
Pepto'.e  blood,  235 
Peptone-plasma,  171 
Peptozym,  234 
Percaglobulin,  510 
Perch  eggs,  66,  504,  509,  510 
Pericardial  fluid,  257,  260 
Perilymph.  494 
Peritoneal  fluid,  257,  261 
Permeability,  of  the  blood-corpuscles,  195, 
196 
of  the  vascular   walls,  257, 

258 
of  the  muscles,  465 
Peroxidases,  17,  18 

See   also    the  tissues  and 
fluids. 
Peroxyproteic  acid,  31 
Perspiratio  insensibilis,  717 
Perspiration,  692-695 
Pettenkofer's  test  for  bile-acids,  135,  312, 
652 
respiration  apparatus,  712, 
Phacozymase,  493 
Phaseomannite,  458 
Phenaceturic  acid,  588,  634,  635 
Phenol-glucuronic  acid,  590,  610,  638 
Phenol-svilphuric  acid  in   the   urine,    589, 
592,  637 
in  sweat,  594 
Phenols,  elimination  by  the  urine,  401,  588 
-591,  633,  637,  638 
in  starvation,  404 
,  estimation   in   urine,    590,    591 
,  formation   in   putrefaction,   30, 
401,  588 
behavior  in  the  animal  body, 
401,  402,  588,  589,  637,  638 
Phenylacetic  acid,  formation  in  putrefac- 
tion, 30,  401 
,  behavior    in   the   ani- 
mal bodv,  588,  634, 
635 
Phenylalanine,  33,  91 

,  behavior    in    the    animal 

body,  5S5,  633 
,  in  alcaptonuria,  597-599 
Phenylaminoacetic  acid,  behavior   in  the 
animal  body,  634 


Phenylaminopropionic  acid,  91 
Phenylbutyric  acid,  634 
Phenylglucosazone,  107,  117 
Phenylhydrazine  test,  107,  117 

in  the  urine,  657 
Phenylketopropionic  acid,  633,  634 
Phenyllactic  acid,  598,  633,  635 
Phenyllactosazone,  524 
Phenylpropionic  acid,     behavior    in    the 

animal  body,  585,  634 
Phenvlpropionic  acid,  formation    in    pu- 
trefaction, 30,  401,  585 
Phenylvalerianic  acid,  634 
Plilebin,  196 
Plilorhizin,  poisoning  with,  282,  297,  610, 

670 
Phlorhizin  diabetes,  297,  610 
Phloroglucin  as  reagent.  111,  374,  666 
Phosphate  calculi,  681 
Phosphates   in  urine,   543,  619-622,  640, 
677-682 
.     See  also  the  different  phos- 
phates   143    144 
Phosphatides,  146,  325,  328^  480,  481,  488 
Phosphaturia,  620 
Phosphocarnic  acid,  454,  457 

in  the  milk,  523, 

532 
in  blood,  186 
in  brain,  480,  481 
in  the  urine,  614 
in    relation    to    the 
elimination  of  CO, 
and    lactic    acid, 
462 
in  relation  to  muscu- 
lar  activity,   470, 
474 
Phosphoglucoproteid,  71,  509 
Phosphoric  acid,  elimination  by  the  urine, 
617-622,  625,  628 
,  formation    in    muscular 

activity,  470 
,  quantitative    estimation 
of,  620-622 
Phosphorized  combinations  in  the  urine, 

614 
Phosphorus  poisoning,  action  on  the  elim- 
ination   of    am- 
monia,   554 
,  action  on  the  elim- 
ination of  urea, 
548,  554 
,  action  on  the  elim- 
ination of  lactic 
acid,    460,    462, 
608 
,  action    upon     the 

blood,  172,  175 
,  fatty  degeneration 
caused  by,  282, 
283,  443 
,  liver  autolysis  in, 
23,  283,  284,  285 


GENERAL  INDEX. 


803 


Pnosphorus  poisoning,  change  in  the  urine. 
285,  460,  54S 
554,  613 

Photomethaemoglobin,  205 

Plirenin,  486 

Phrenosin,  483,  485,  488 

Phthalic  acid,  behavior  in  the  body,  fi33 
from  choHc  acid,  315 

PhthaUmide  malonic  ester,  102 

PhyUocyanin,  214 

Phylloporjihyrin,  197,  214 

Phylloerythrin,  324 

Phymatorusin,  688 

in  the  urine,  651 

Physetoleic  acid,  138 

Pliysiological  availability,  727 

Phytosterines,  335 

a-Picoline,  behavior  in  the  animal  body, 
637 

Picric  acid,  reagent  for  protein,  42,  646 
,  reagent     for   creatinine,    565 

567 
,  reagent    for  sugar,   117,    56G 

Pigment  calculi,  334 

Pigments  of  the  eye,  489-491 
of  the  blood,  196-219 
of  the  blood-sermn,   186,   J8f- 

505 
of  the  corpora  lutea,  216,  49S 
of  the  egg-shell,  509 
of  feathers,  690 
of  the  fat-cells,  441 
of  the  bile,  319-325,  328,  331 
of  the  urine,  600-  607 
of  the  skin,  688-691 
of  the  lobster,  509,  690 
of  the  liver,  282 
of  the  muscles,  453,  454 
of  lower  animals,  218,  219 
,  medicinal  pigments  in  the  urine, 
639,  654 

Pigmentary  acholia,  329 

Pig's  milk,  529 

Pike,  flesh  of,  476 

Pilocarpine,  action  on  the  secretion  of  in- 
testinal juice,  377 
,  action  on  the  elimination  of 

CO^  in  the  stomach,  372 
,  action    on    the    secretion    of 

saliva,  347 
,  action  on  the  elimination  of 
uric  acid,  569 

Piqure,  299 

Piria's  tyrosine  test,  90 

Placenta,  512 

Plant  gums,  128,  129 

Plant  nucleic  acids,  156 

Plants,  chemical  processes  in,  1,  2 

Plasma.    See  Blood-plasma. 

Plasminic  acid,  156 

Plasmoschisis,  227 

Plasmozym,  232 

Plastein,  56,  363,  396 

Plasteinogen,  57 

Plastin,  149 


Plattner's  cr^'stallized  bile,  311 

Plethora  polycythaimia,  245 

Pleural  iiuid,  257,  261 

Plums,  action  on  the  elimination  of  hip- 

puric  acid,  585 
Pneumonic  infiltration,  solution  of,  23, 268, 

713 
Poikilocytosis,  247 
Polarization  test,  664 
Polycythicmia,  245,  248 
Polypeptides.    See  Peptides. 
Polyperythrm,  691 
Polysaccharides,  126 
Polyuria,  628 
Pons  varolii,  486 

Poorhouses,  diet  of  inmates  of,  770 
Pork,  475 

Pork-fat,  absorption  of,  423 
Portal  vein,  blood  of,  241,  295,  296,  419 
Positive  phase,  234 

Potassium  combinations,  division     of,    in 
the   form-ele- 
ments      and 
fluids,       166, 
167,  464 
,  elimination     of, 
in  fevers,  623 
,  elimination      of 
in  starvation, 
623,  731 
,  elimination     by 
the  saliva,  847 
in  the  urine,  623 
Potassiiim  chlorate,  poisoning  with,  203 
Potassimn  phosphate  in  yolk  of  eggs,  505 
in  muscles,  464,  465, 

477 
in    cells,    166,    167, 

168 
in  spermatozoa,  496 
Potassium  sulphocyanide  in  the  urine,  611 
in    saliva,   341, 

343 
in  gastric  con- 
tents, 354 
Potatoes,  absorption  of,  in  the  intestine, 

421 
Potential  energy  of  various  foods,  724-728 
Precipitins,  186,  413 
Preglobulin,  141,  228,  271 
Preputial  secretion,  692 
Primary  proteoses,  52 
Prisoners,  food-ration  for,  770 
Proliferous  cvsts,  498 
ft-Proline,  34",  74,  101 
Prolineglycyl  anhydride,  35 
Propepsin,  364 
Prope}>tones,  50 
Propylalanine.  35 
Propyl  benzene,  behavior  in  the  animal 

body,  634 
Propylene  glj'^col,  relation  to  formation  of 

glycogen,  291 
Prosecretin,  379,  384 
Prostatic  calculi,  498 


804 


GENERAL  INDEX. 


Prostatic  secretion,  496 
Prosthethic  group,  71 
Protagon,  148,  271,  480,  481,  482 
Protalbinic  acid,  49 
Protojiroteoses,  51 

Protamines,  36,  58,  62,  63,  149,  497,  498 
.Proteids,  36,  37-65. 

,     See   also   tlie  various    protein 
groups. 
Protein,  separation    from    fluids,    44 

,  approximate    estimation    in    the 

urine,  646 
,  circulating    and    tissue    protein, 

741-745 
,  action  on  the  formation  of  gly- 
cogen, 292,  294,  303-305 
,  active,  4 

,  living  and  non-living,  4 
,  detection  and  quantitative  esti- 
mation of,  43-45,  639-646 
,  regeneration  of,  415,  416,  417 
,  absorption  of,  412-418 
,  passage  of,  into  the  urine,  640 
,  heat  of  combustion  of,  723-726 
,  digestibility  in  gastric  juice,  356, 

368 
,  digestibility  in  pancreatic  juice, 

393 
,  formation  of  sugar  from,  303-305 
Protein  bodies  in  general,  26-83 

,  summary  of  the  various, 

36,  37 
.    See  also  the  various  pro- 
tein bodies  of  the  tissues 
and  fluids. 
Protein  hydrogele,  39,  40 
Protein  hydrosole,  39,  40 
Protein  content  affected  by  inoculation, 

188 
Protein  metabolism  in  work  and  rest,  471- 
475,  758  _ 
in    starvation,     728, 

729 
in  various  ages,  757, 

758 
with  different  foods, 

739-745 
ante-mortem  increase, 

730 
after     feeding     with 
thyroid      extracts, 
276  _ 
Protein  overfeeding,  751,  752 
Protein  putrefaction,   30,   401-408,   585, 

588,  589 
Protein,  relation  to  the  albuminates,  49 
Proteincystine,  92 
Proteinochromogen,  30,  102 
Protein  substances,  26-82 

,  synthesis  of,  34,  35 
,  action  upon  coagula- 
tion  of  the  blood, 
233 

See  also  individual 
protein  bodies. 


Proteoses,  36,  52 

,  general  properties  and  prepar- 
ation, 50-61 
in  blood,  183,  247,  415 
,  formation  in  protein  putrefac- 

tion,  50,  401,  415,  416 
,  relationship  to  the  coagiilation 
of  the  blood,  171,  226,  234, 
235 
,  nutritive  value,  745,  746 
,  absorption  of,  414-417 
,  transformation  into  protein,  415 
,  occurrence  in  urine,  643 
Prothrombin,    176,    231,   232,   233 
Protic  acid,  454 
Proteolytic  enzymes,  16,  263 
Protocatechuic  acid,  behavior  in  the  body, 

591 
Protoelastose,  76 
Protogelatose,  79 
Protogen,  49 
Protokyrin,  58 
Protones,  63 
Protoplasm,  4,  140,  141 

and    protein    decomposition 
of,  548 
Protosyntonose,  100 
Pseudocerebrin,  485 
Pseudochvlous  fluid,  262 
Pseudoglobulin,  179,  643 
Pseudoglycogen  formers, 
Pseudohsemoglobin,  203  293 
Pseudolevulose,  108 
Pseudomucin,  68,  500 

in  ascitic  fluids,  262 
in  cysts,  500 
in  the  gall-bladder,  329 
Pseudonucleins,  47,  151,  359, 

from  casein,  521,  531 
from  vitellin 
Pseudopepsin,  354 
Pseudotagatose,  108 
Pseudoxanthine,  457 
Psittacofulvin,  690 
Psylla-alcohol,  692 
Psyllic  acid,  692 

Psychical  period  of  secretion,  350 
Ptomaines,  24 

in  the  urine,  615,  676 
Ptyalin,  343,  344 

,  behavior  with  acid,  345,  366 
,  action  on  starch,  344-346 
,  tests,  345,  346 
Pulmotartaric  acid,  713 
Purine,  157 
Purine  bases,  156,  578 

,    See  also  Nuclein  bases. 
Purple,  691 
Purple  cruorin,  202 
Pus,  266-269 
,  blue,  269 
cells,  267 
in  urine,  651 
corpuscles,  267 
serum,  266 


GENERAL    INDEX. 


805 


Putrefactive  processes,  2.5,  30 

in   intestine,  400- 
40S,    585,    588- 
596 
Putrescine,  24,  97 

in  intestine,  24,  676 
in  the  urine,  615,  676 
Pyin,  261,  266,  269 
Pyinic  acid,  269 
Pyloric  gland,  348 
Pyloric  secretion,  365 
Pyocyanin,  269 

in  sweat,  695 
Pyogenin,  268,  483 
Pyosin,  268,  483 
Pyoxanthose,  269 

Pyridine,  beliavior  in  the  body,  639 
a-Pyridine-carboxylic  acid,  632,  636 
Pyrocatechin,  591 

,  occurrence  in  urine,  591 
,  occurrence  in  transudates, 
259,  265 
Pyrocatechin-sulphuric  acid,  591 
Pyromucic  acid,  637 
Pyromucinornithuric  acid,  637 
Pyrrol  derivatives,  210,  689 
a-Pyrrolidinecarboxyhc  acid,  101-103, 197. 

See  «-Proline. 
Pyrrolidonecarboxylic  acid,  74 

Quercite,  relation  to  glycogen  formation, 

291 
Quinic  acid,  behavior  in  the  animal  body, 

586 
Quinine,  passage  of,  into  urine,  639 

,  passage  of,  into  sweat,  695 

,  action  of,  on  the  elimina.tion  of 

uric  acid,  569 
,  action  on  the  spleen,  274 
Quotient,  respiratory,  306,  446,  472,  722 

731,  760 
Quotient,  urea  to  nitrogen,  628,  720 
,  nitrogen  to  sugar,  304,  306 

Racemic  acid,  behavior  in  the  animal  body, 

539 
Rachitis,  bones  in,  438,  440 
Rape-seed  oil,  feeding  with,  442 
Reductases,  19 
Reduction  processes,  2,  5,  18,  19.    See  also 

the  various  chapters. 
Reichert-Meissl's  equivalent,  137 
Reindeer,  milk  of,  529 
Rennin,  25,  56.     See  also  Chymosin. 
Rennin  cells,  349 
Rennin  glands.  349 
Rennin  zymogen,  349,  361.  362 
Reproductive  organs,  419-513 
Resacetophenon,  637 
Residual  nitrogen,  183 
Resin  acids,  transition  into  urine,  639,  641 
Respiration,  anaerobic,  21,  461 
,  external,  696,  703 
,  internal,  696,  703,  712" 
of  the  hen's  egg,  511,  512 


Respiration  of  plants,  2 

See  also  Chemistry  of  res- 
piration,   696-714,    and 
Exchange    of   gas  under 
various  conditions. 
Respiratory  quotient,  306,  446,  472,  722, 

731,  760 
Rest,  metabolism  during,  467-472,  758- 

761 
Reticulin,  37,  80,  428 
Retene,  335,  336 
Retina,  489 
Reversion,  124 
Revertose,  16 

Reynolds'  acetone  reaction,  671 
Rhamnose,  relation  to  glycogen  formal  ion, 

290,  336 
Rheometer,  706 
Rhodizonic  acid,  458 
Rhodophan,  491 
Rhodopsin,  489 

Rhubarb,  action  on  the  urine,  639 
Rib-cartilage,  434 
Rigor  mortis  of  the  muscles,  465 
Roberts'  method  of  estimating  sugar,  663 
Roch's  reaction  for  protein,  642 
Rodents,  bile-acids  of,  318 
Rods  of  the  retina,  pigments  of,  490 
Rosenbach's  bile-pigment  test,  852 

urine  test,  675 
Rotation,  specific,  109 
Rosin's  levulose  reaction,  119,  665 
Rovida's  hyaline  substance,  141,  221,  267 
Rubner's  sugar  test,  117,  665 
Rye  bread,  utilization,  417,  421,  727 

Saccharic  acid,  106,  107,  121 
,  lactone  of,  121 
,  relation    to  glycogen  for- 
mation, 291 
,  behavior  in  diabetes,  300 
Saccharose,  123,  124 

calorific  value,  724 
absorption  of,  419 
Salicylase  or  aldehydase,  18 
Salicylic  acid,  action  on  pepsin  digestion, 
359 
,  action  on  trypsin  digestion, 

393 
,  behavior    in     the    animal 
body,  635 
Salicylic-acid  amyl  ester,  284 
Salicylsulphonic  acid  as  protein  reagent, 

42 
Saliva,  339-348 

,  secretion  of,  346,  347 

,  mixe4,  342 

,  physiological  importance,  348 

,  behavior    in    the    stomach,    348, 

366,  367 
,  action  of,  345,  348,  367 
,  gases  of,  340,  702 
,  composition  of,  346,  347 
Salivary  calculi,  348 
Salivary  diastase.     See   Ptyalin. 


806 


GENERAL  INDEX. 


Salivary  glands.  339 

Salkowski's  cholesterin  reaction,  336 

Salkowski-Ludwig's  method  of  estimating 

uric  acid,  336 
Salmine,  63,  64 
Salmon,  flesh  of,  454 

,  sperma  of,  63,  155,  497 
Salmonucleic  acid,  154 
*Salts,  action  of,  upon  metabolism,  754 
,  antagonistic  action  of,  377,  375 
.     See  also  the  various  salts. 
Salt-plasma,  171 
Salts  of  vegetable  acids,  behavior  in  the 

organism,  544 
Samandarin,  692 

Santonine,  action  on  the  urine,  639,  654 
Sapokrinin,  385 
Saponification  equivalent,  136 
Saponification.  133,  388,  398,  422,  423 
Saponin,  193,  337 

Sarcolactic  acid.     See  Paralactic  acid. 
Sarcolemma,  447 
Sarcomelanin,  688 
Sarcomelanic  acid,  688 
Sarcosine,  455 

,  behavior  in  the  animal  body, 
630 
Sarkine.     See  Hypoxanthine. 
Scherer's  inosite  test,  459 
Schiff's  reaction  for  cholesterin,  336 
reaction  for  uric  acid,  576 
reaction  for  urea,  555 
Schreiner's  base,  496 
Schiitz-Borissow's  law,  390,  392 
Schweitzer's  reagent,  107 
Sclerotic,  493 
Scombrine.  59,  63,  64 
Scombron,  62 
Scyllite,  272 
Scymnol,  310 

Scymnol-sulphuric  acid,  310 
Seal-fat,  138 
Sea-urchin,  sperm  of,  62 
Sebacic  acid,  135 
Sebum,  691 

Secondary  proteoses,  52 
Secretin,  309,  377,  379,  384 
Secretin  enzymes,  22 
Sediments.    See  Urinary  sediments. 
Sedimentum  lateritium,     543,    575,  607, 

677 
Seliwanoff's    reaction   for    levulose,    119, 

665 
Semen,  495-497 

Semicarbazide,  poisoning  with,  584 
Semiglutin,  79 
Seminose.    See  Mannose. 
Senna,  action  on  the  urine,  639,  654 
Sepsine,  24 
Seralbumin,  36,  181 

detection  of,  in  the  urme,  640, 
643 
,  quantitative    estimation    of, 

183,  645 
,  absorption  of,  413 


Serglobulin,  36,  178 

,  detection  of,  in  the  urine,  640, 

643 
,  quantitative    estimation    of, 
180,  645 
Sericin,  37,  82 
Serine,  33,  95 
Serolin,  183 
Serosamucin,  258 
Serous  fluids,  256-265 
Serum.     See  Blood-serum. 
Serum  casein.     See  Serglobulin. 
Sex.  influence  on  metabolism,  756 
Sharks,  bile  of,  310,  325 

,  urea  in.  240,  325,  547 
Sheep's  milk,  529 
Shell-membrane    of    the    hen's    egg,    73, 

509 
Silicic  acid  in  feathers,  685 
in  hair,  685 
in  urine,  626 

in  hen's  egg,  505,  509,  510 
in  connective  tissue,  429 
Silicic  acid  ester  in  feathers,  685 
Silk  gelatine,  82 
Sinistrin,  animal,  71 
Silver,  in  blood,  239 
Skatol,  29,  34,  102,  401,  402 

,  formation  in  putrefaction,  29,  401, 

588 
,  behavior  in  the  animal  body,  400, 
401,  588,  595,  634 
Skatolacetic  acid,  30,  102 
Skatolaminoacetic  acid,  30,  102 
Skatolcarboxylic  acid,  102,  596 
Skatol-pigment,  595,  596,  607 
Skatosine,  103 
Skatoxyl,  401,  588,  634 
Skatoxylglucuronic  acid,  595 
Skatoxylsulphuric  acid,  588,  595 

in  sweat,  694 
Skeletins,  81 

Skeleton  at  various  ages,  438 
Skin,  685-695 

,  excretion  through,    690,    692-695, 
717 
Sleep,  metabolism,  761 
Small  intestine,  378,  380 

extirpation  of,  427 
Smegma  prseputii,  692 
Smith's  reaction  for  bile-pigments,  654 
Smooth  muscles,  477 
Snail  mucin,  66 
Snake   poison,    action  upon    blood,   193, 

226,  234 
Soaps  in  blood-serum,  183 
in  chyle,  251,  423 
in  pus,  268 

in  excrements,  408,  426 
in  bile,  310,  325 
in  milk,  532 
,  imi^ortance  of,  in  the  emulsification 
of  fats,  389,  390,  398,  423 
Sodium    alcoholate    as    a  saponification 
agent,  136,  659 


GENERAL  INDEX. 


807 


Sodium  cliloride,  elimination  by  the  urine, 
616,  617,  694,  695 
,  elimination  by  the  sweat, 

694,  695 
,  physiological  i  m  p  o  r  t- 

ance,  736 
,  quantitative  estimation, 

616-619 
, influence  on  the  quan- 
tity of  urine,  754 
,  influence  on  the  elimina- 
tion of  urea,  754 
,  influence  on  the  secre- 
tion of  gastric  juice, 
364,  736 
,  behavior  with  food  rich 

in  potassium,  736 
,  insufficient     supply    of, 

364,  736 
,  action  on  the  secretion 
of  intestinal  juice,  377, 
378 
,  action  on  pepsin  diges- 
tion, 358,  359 
,  action  on  trypsin  diges- 
tion, 393,  394 
Sodium  compounds,  elimination    by    the 
urine,  623 
,  division    among    the 
form-elements  and 
fluids,  166 
.    See  also  the  vari- 
ous    tissues      and 
fluids. 
Sodium    phosphate    in    the    urine,    619, 

620 
Sodium  salicylate,  action  on  the  secretion 

of  bile,  309 
Sodium    tartrate,    relation    to    glycogen 

formation,  291 
Solanin,  193 
Soldiers,  diet  of,  769 
Sorbin,  119 
Sorbinose,  113,  119 
Sorbite,  106 

Source  of  mviscular  energy,  472-474 
Spawn  of  tlae  frog,  71 
Specific  rotation,  109 
Spectrophotometry,  218 
Sperma,  63,  495-498  ' 
Spermaceti,  138 
Spermaceti  oil,  138 
Spermatin,  498 
Spermatocele  fluids,  263 
Spermatozoa,  497 
Spermine,  496 
Spermine  crystals,  496 
Spherules,  37,  503,  509 
Sphingosin,  485 
Sphygmogenin,  278 
Spider  excrement,  guanin  therein,  160 
Spiegler's  reagent,  642 
Spirographin,  69 
Spirogyra,  114,  167 


Spleen,  272-275 

,  relation  to  formation  of  blood,  274 
,  relation  to  formation  of  uric  acid, 

274,  570,  573 
,  relation  to  digestion,  385 
,  blood  of  the,  242 
Spleen  pulp,  272 
Splitting  processes  in  general,  1,  2,  9.    See 

also  the  various  enzymes. 
Spongin,  37,  81,  82 
Sputum,  714 
Sputum  mucin.     See  Mucin  from  mucous 

membrane,  66 
Starch,  126 

,  hydrolytic  cleavage  by  diastase, 

128,  387,  388 
,  hydrolytic  cleavage  by  pancreatic 

diastase,  388 
,  hydrolytic  cleavage  by  saliva,  344 
,  calorific  value,  724 
,  absorption,  419,  421 
Starches,  digestion  of,  367,  388 
Starch,  cellulose,  126 
Starch  granulose,  126 
Starvation,  action  on  the  blood,  244,  731, 
732 
,  action  on  the  urine,  404,  548, 

585,  592 
,  action  on  the  elimination    of 

indican,  404,  592 
,  action  on  the  elimination    of 

oxalic  acid,  582 
,  action  on  the  secretion  of  bile, 

307,  308 
,  action   on    the    secretion    of 

pancreatic  juice,  382 
,  action  on  the  elimination  of 

phenol,  404 
,  action    on    metabolism,    722, 

728-733 
,  death  from,  728 
Starvation  cures,  770,  771 
Starvation  requirement,  733,  755 
Steapsin,  388 
Stearic  acid,  133 
Stearin,  133 

,  absorption  of,  423 
Stentorin,  blue,  691 
Stercobilin,  320,  409,  603 
Stercorin,  337 
Stethal,  138 

Stokes's  reduction  fluid,  203 
Stokvis'  reaction  for  bile-pigments,  653 
Stojnach,  gases  in  the,  372 

,  importance  in  digestion,  369 
,  pepsin  charge  in,  364 
,  relation  to  intestinal   putrefac- 
tion, 371,  406,  407 
,  auto-digestion  of,  372 
,  digestion  in  the,  365-372 
Stomachic  glands,  350 
Stone-cystine,  92 

Streptococcus,  behavior  with  gastric  juice, 
371 


808 


GENERAL  INDEX. 


Stroma  fibrin,  195 
Stroma  of  the  blood-corpuscles,  194 
of  the  muscles,  451 
of  the  ovaries,  498 
Strontium  salts  and  blood    coagulation, 

171 
Struma  cystica,  275 
Strychnine,  passage  of,  into  the  urine,  639 

and  sugar  elimination,  299 
Sturgeon,  sperma  of,  63 
Sturine,  63,  100 
Sublingual  glands,  339 
Sublingual  saliva,  341 
Submaxillary  glands,  339 
Submaxillary  mucin,  66,  67 
Submaxillary  saliva,  340,  341 
Succinic  acid  in  putrefaction,  32 

in  the  fermentation  of  milk, 

516 
in  the  intestine,  400 
in  the  spleen,  272 
in  transudates,  259,  264 
in  the  thyroid  glands,  275 
from    phosphocarnic    acid, 
457 
,  passage  of,  into  the  urine, 

630 
,  passage  of,  into  the  sweat, 
695 
Sugar,  relation  to  work,  469,  473 

,  formation  from  fats,  306,  473 
,  formation  from  protein,  303-305 
Sugar  formation,  in    the    liver,    295-301, 
306 
after  pancreas  extirpa- 
tion, 301-305 
Sugar,  behavior     on     subcutaneous     in- 
jection, 293 
,  behavior  to  blood-corpuscles,  195 
,  quantitative    determination,    659- 
665 
.    See  also  various  kinds  of  sugar. 
Sugar  tests  in  the  urme   655-659 
Sulphsemoglobin,  207 
Sulphocyanides  in  the  urine,  611,  631 
in  gastric  iuice,  354 
in  the  saliva,  341,  343 
Sulphonal  intoxication,  urine  in,  213,  650 
Sulphonic  acids,  behavior  in  the  animal 

body,  540 
Sulphur,  of  proteins,  27.    See  also  various 
proteins. 
,  in  the  urine,  471,  611,  612 
,  elimination  of,  in  activity,  471 
,  elimination  of,  with  lack  of  oxy- 
gen, 611 
,  neutral  and  acid  sulphur  in  urine, 

611 
,  behavior  in  the  organism,  611, 
631 
Sulphur  methjemoglobin,  207 
Sulphuretted  hydrogen  in  putrefaction  in 
the       intestine, 
401,  404 
in  the  urine,  612 


Sulphuric  acid,  ethereal  and  sulphate,  in 
the  urine,  588,  589,  611, 
622,  623 
,  elimination  of,  in  activity, 

471 
,  elimination     of,     by    the 
urine,  543,  622,  623,  628 
,  elimination     of,     by     the 

sweat,  694 
,  estimation  of,  622 
,  relation  to  elimination  of 

nitrogen,  471,  611,  622 
,  action  on  pepsin  digestion, 
358 
Suprarenal  capsule,  277 
Suprarenin,  278.    See  also  Adrenalin. 
Swallow's-nests,  edible,  69 
Sweat,  692-695 

Swimming-bladders  of  fishes,  gases  of, 7 10 
,  guanine    in, 
160 
Sympathetic  saliva,  340 
Synproteose,  55 
Synovia,  265 
Synovial  fluid,  265 
Synovial  mucin,  258 
Synoviamucin,  265 
Synovin,  266 
Synthesis,  1,  2, 

of  ethereal  sulphuric  acids,  280, 
401, _  588,  590,  593,  594,  637 
of  conjugated  glucuronic  acids, 
122,  589,  593,  609,  610,  632, 
638 
of  uric  acid,  567,  568,  572,  573 
of  urea,  547,  550,  551,  552 
of  hipinu'ic  acid,  3,  585,  635 
of  varieties  of  sugars,  106,  114 
of  polypeptides,  36, 
Syntonin,  48,  100 

,  calorific  value  of,  726 

Tagatose,  108 

Talonic  acid,  120 

Talose.  108,  113,  120 

Tapeworm  cysts,  265 

Tannic  acid,  behavior  in  the  animal  body, 

637 
Tartar,  348 

Tartaric  acid,  relation  to  glycogen  forma- 
tion, 291 
,  passage  of,  into  sweat,  695 
,  behavior     in     the     animal 
body,  630 
Tartronic  acid,  573 
Tatalbumin,  506 
Taurine,  94,  95,  310,  313,  331 

,  behavior  in  the  animal  body,  629 
Taurocarbamic  acid,  631 
Taurocholeic  acid,  314 
Taurochohc  acid,  310,  313,  328 

,  occurrence  in  meconium. 


410 

,  decomposition     in 
intestine,  404 


the 


GENERAL   INDEX. 


809 


Taurocholic  acid,  protein-precipitating  ac- 
tion, 42,  647 
Tea,  action  on  metabolism,  755 
Tears,  418 
Teeth,  440 

Teichmann'i  crystals,  211,  649 
Tendon  mucin,  66 
Tendon  mucoid,  428 
Tendon  svnovia.  265 

Tension  of  the  CO.  in  the  blood,  708-710 
in  the  tissues,  712 
in  the  Ivmph,  251 
O   in  the  blood,  703-708 
Terpen-glucuronic  acid,  667 
Terpentine,  action  of,  on  the  secretion  of 
bile,  308 
,  action  of,  on  the  urine.  639 
,  behavior  in  the  animal  bodv, 
609,  638 
Tetraglycylglycine,  35,  396 
Tetraoxj^aminocaproic  acid,  96 
Tetrapeptides,  35,  395 
Tetronerj'thrin,  219,  690 
Testes,  495 
Tetroses,  105 
Theobromine,  157 

,  behavior     in     the     animal 
body,  579 
Theophylline,  157 

,  behavior     in     the     animal 
body,  579 
Thioalcohols,     behavior    in    the    animal 

body,  632 
Thioglycolic  acid,  74 

,  behavior  in  the  animal 
bodv,  632 
Thiolactic  acid,  28,  33,  94 
Thiophene,  behavior  in  the  animal  body, 

637 
Thiophenic  acid,  637 
Thiophenuric  acid,  637 
Thiotolene,  637 

Thrombin,  13,  16,  175,  228,  233 
Thrombogen,  231-233 
Thrombokinase,  231 
Thrombosin,  229 
Thymine,  152,  154,  165 
Thymic  acid,  154 
Thymonucleic   acid,   151,    152,   153,    154, 

155 
Thymus,  270 
Thyreoglobulin,  276,  277 
Thyreoidea,  275-277 
Thyreoproteid,  276 
ThjTeotoxalbumin.  277 
Thyroid  gland,  275,  276 
Thyroiodin.     See  lodothyrin. 
Tissue-fibrinogen,  141,  271 
Tissue  proteids. 

ToUens'  reaction  for  pentoses,  111,  112 
Toluene,  behavior  in  the  animal  bodv,  585, 

634  _ 

Toluric  acid,  635 

Toluylenediamine,  poisoning  with,  333  ' 
Toluic  acid,  635 


Tonas,  chemical  of  the  muscle,  467 

Tooth  structure,  440 

Tortoise,  bones  of,  436 

Tortoise-shell,  73,  691 

Toxalbumins,  behavior  with  gastrin  juice, 

371 
Toxines,  24,  25,  186,  2S0 
Tracheal  cartilage,  420,  433 
Transudates.  256-266 
Tribromacetic  acid. 
Tricalcium  casein.  520 
Tricliloracetic  acid  as  reagent,  42,  45 
Trichlorethyl-glucuronic  acid.     See  Uro- 

cliloralic  acid. 
Triglycylglycine,  35,  396 
Triolein,  34 
Trioses,  105 
Tripalmitin,  1.34 
Tripeptides.  35,  .395 

Triple  phosphate  in  urinary    sediments, 
678,  679 
in  urinars'  calculi,  68J, 
681,  682 
Tristearin,  133 
Triticonucleic  acid,  152,  156 
Tronmier's  test  for  sugar,  116,  655 

,  behavior     with 
uric  acid,  575 
,  behavior     with 
creatinine, 
565 
Tropics,  metabolism  in  inhabitants  of,  762 
Trypsin,  186,  390-395 

,  action  on  proteins,  54.  .55,  392, 416 
,  action  on  peptides,  35,  .395.  396 
,  importance  in  absorption,  416 
Tr>T)sin  digestion,  392-396 

,  products  of,  394 
Trj-psinogen,  383-386 
Trvqjsin  peptone,  51,  54,  55,  57,  58 
Tryptophane,  30,  102,  355 
Tubo-ovarial  cysts,  501 
Tunicin,  685 
Turacin,  690 
Turacoverdin,  690 
Trj-osine,  18,  33,  54,  89-91 
,  in  urine.  675 
,  in  sediments,  680 
,  detection  of,  91,  675 
,  behavior   in  putrefaction,   400, 

585,  588 
,  behavior  in  the  animal   body, 
598,  599,  633,  670 
Trj'osinases,  18,  90,  690 
Tyroslne-sulphuric  acid,  90 

UfTelmann's  reaction  for  lac  ic  acid,  374 
Umikoff's  reaction.  532,  533 
Uracil,  152,  156.  164 
Uraemia,  bile  in,  329 

,  gastric  contents  in,  373 

,  sweat  in,  694 
Uraminobenzoic  acid,  636 
Urates,  542 

,  in  sediments,  575,  678 


810 


GENERAL  INDEX. 


Urea,  547 

,  elimination  in  starvation,  548 

,  elimination  in  children,  549,  665 

,  elimination  in  disease,  547,  548,  554 

,  properties  and  reactions,  554 

,  formation  and  origin,  550-554,  630 

,  quantitative  estimation,  558-562 

,  synthesis,  547,  551-553 

,  occurrence  in  the  blood,  186,  240, 

242,  551 
,  occurrence  in  the  bile,  325,  329,  547 
,  occurrence  in  the  liver,   547,   550, 

551 
,,occurrence  in  the  muscles,  455,  547 
,  occurrence  in  transudates,  259 
Urea  glucuronic  acid,  609 
Urea  nitrate,  555 
Urea  oxalate,  555 
Ureides,  30,  568,  583 
Urein,  562 
Urethane,  563 
Urease,  16,  677 

Ureido-gluciu-onic  acid,  609,  611 
Uric  acid,  157,  550,  567,  568 

,  elimination  in  disease,  570 

,  elimination    after  feeding  with 

nuclein,  569,  571 
,  relation  to  urea,  567-573 
,  properties  and  reactions,  573- 

576 
,  formation  in  the  animal  body, 

570-574 
,  quantitative  estimation,  576- 
.  578 

,  syntheses  of,  568,  572,  573 

,  behavior  in  the  animal  body, 

573,  574 
,  occurrence  of,  568,  569 
,  occurrence  of,  in  sweat,  547,  694 
,  occurrence  of,  in  sediments,  543, 
575 
Uric-acid  calculi,  681 
Urinary  calculi,  680-683 
Urinary  pigments,  601-607 

,  medicinal,  639,  654 
Urinary  sand,  680 

Urinary  sediments,  542,  543,  677-680 
Urine,  541-684 

,  excretion  of,  626,  627 

,  inorganic  constituents  of,  616-626 

,  poisonous  constituents  of,  615 

,  organic    pathological    constituents 

of,  639-676 
,  physiological  constituents  of,  546- 

616 
,  enzj^nes  of,  615 
,  casual  constituents  of,  629-639 
,  color  of,  542,  601,  628,  639,  648-654 
,  solids,  calculation  of,  626,  627 
,  quantity  of  solids,  626,  627 
,  alkaline  fermentation  of,  677 
,  acid  fermentation  of,  677 
,  gases  of,  626,  703,  712 
,  quantity  of,  626,  627 
,  physical  properties  of,  542-547 


Urine,  osmotic  pressure  of,  546 

,  physico-chemical  analysis  of,  628 

,  reaction  of,  543-546 

,  acidity  of,  543-545 

,  estimation  of  acidity,  545 

,  specific  gravity  of,  546,  627,  628 

,  passage  of  foreign  bodies  into,  629- 

639 
,  composition  of,  628 
,  reducing  power  of,  608 

Urine  indican,  592 

Urine  indigo,  592,  601 

Urine  poison,  615 

Urine  purines,  endogenous  and  exogenous, 
_  578-581 

Urine  sugar.     See  Dextrose. 

Urinometer,  546 

Urobilin,  601,   602-607  _ 

,  relation  to  bilirubin,  319,  332, 

404,  603 
,  relation  to  choletelin,  603 
,  relation   to    hsematin,   332,    603 
,  relation    to     hsematoporphyrin, 

214,  603 
,  relation   to  hydrobilirubin,  332, 
603 

Urobilin  icterus,  604 

Urobilinogen,  601,  605 

Urobilinoid  bodies,  603 

Urocamic  acid,  526,  615 

Urocliloralic  acid,  122,  632 

Urochrome,  601,  602 

Urocyanin,  601 

Uroerythrin,  601,  607 

Uroferric  acid,  611,  613 

UrofvLscohsematin,  650,  651 

Uroglaucin,  601 

Urohfematin,  601 

Urohodin,  601 

Uroleucic  acid,  597,  60 

Uromelanins,  601 

Uronitrotoluolic  acid,  638 

Urophsein,  601 

Uroproteic  acid,  613 

Urorubin,  601 

Urorubrohsematin,  650 

Urorosein,  596,  601,  651 

Urospectrin,  650 

Urostealith  calculi,  682 

Urotheobromine,  580 

Urotoxic  coefficient,  615 

Uroxanthine,  592 

Uroxonic  acid,  568 

Ursocholeic  acid,  319 

Uterine  milk,  512 

Uterus  colloid,  502 

Utilization  of  the  various  foodstviffs,  417, 
421,  425,  530,  531 


Valerianic  acid,  29 

Vegetable   acids,   behavior   of   the   alkali 

salts  of,  in  body,  544 
Vegetable  gums,  120,  129 
Vegetable  mucilages,  128,  129 


GENERAL  INDEX. 


811 


Vegetarians,  food  of,  750,  767 

,  excrement,  408 
Vernix  caseosa,  336,  691 
Vesicatory  blisters,  265 
Vesicle  calculi, 
Vesiculase,  496 
Virtual  sugar,  184 
Visual  purple,  489-491 
Visual  red,  489 
Vitali's  pus-blood  test,  649 
Vitellin,  36 

in  yolk  of  egg,  503 

in  protoplasm,  141 
Vitellolutein,  505 
Vitellorubin,  505 
Vitelloses,  52 
Vitreous  humor,  491 

"Water,  drinking  of,  action  in  the  elimina- 
tion   of     chlorides, 
616 
,  action  on  the  elimina- 
tion   of    uric   acid, 
569 
,  action  on  the  elimina- 
tion of  urea,  753 
,  action  on  the  deposi- 
tion of  fat,  754 
,  action   on   the  excre- 
tion of  urine,  626 
,  elimination  of,  through  the  urine, 

626-628,  717 
,  elimination  of,    through  the  skm, 

693,  717 
,  elimination  of,  in  starvation,  731 
,  elimination  of,  importance  for  the 

animal  body,  734 
,  elimination  of,  quantity  of,  in  the 

various  organs,  734 
,  elimination,  lack  of,  in  the  food, 
734 
Wax,  138 

in  plants,  691 
Weidel's  xanthine  reaction,  160 
Weyl's  reaction  for  creatinine,  565 
Wheat  bread,  absorption  of,  418 
Whey,  516,  528 
Whey  proteid,  521 
White  of  egg,  506-510 

,  calorific  value  of,  724 
,  absorption  of,  413,  414 
Witch's  milk,  534 
Woman's  milk.    See  Human  milk. 


Wool-fat,  337,  692 

Work,     action    on    the    elimination    of 
clilorine,  616 
,  action  on  the  elimination  of  sul- 
phur, 471 

,  action  on  the  excretion  of  nitrogen, 

470,  471 
,  action  of   the   necessity  for  food, 

768,  709 
,  action  on  metabolism,  468-474,  758 
-761 
Worm-Miiller's  sugar  test,  655 
Wound  secretion,  265 

Xanthine,  157,  159 

in  the  urine,  578 
in  urinary  calculi,  682 
in  urinary  sediments,  680 
,  detection  and  quantitative  esti- 
mation,  159,  160,  163,  164, 
580,  581 
Xanthine  bodies.     See  Nuclein  bases. 
Xanthine  calculi,   682 
Xanthine  oxidase,  18,     73,  571 
Xantho-creatinine,  457,  470,  567 
Xantho-melanin,  32 
Xanthophan,  491 
Xanthophyll  groups,  505 
Xanthoproteic  reaction,  42 
Xylene,  behavior  in  the  animal  body,  634 
Xyliton,  689 
Xyloses,  113,  282 

,  relation     to    the    formation    of 
glycogen.  111,  290 

Yeast-cells,  relation  to  fermentation,  10, 

11 
Yeast  maltase,  17 
Yeast  nucleic  acid,  152,  156 
Yeast  nuclein,  150 
Yolk  of  the  hen's  egg,  502-506 
Yolk-spherules,  37,  503,  509 

Zein,  27,  98 

Zinc  in  the  bile,  325 

in  the  liver,  286 

,  passage  of,  into  milk,  539 
Zooervthrin,  690 
Zoofulvin,  690 
Zoorubin,  690 

Zymase,  10,  11,  20,  21,  396 
Zymogens,  15.     See  various  enzymes. 
Zymoplastic  substances,  176,  228,  231 


INDEX  TO  AUTHORS. 


Abderhalden,  E.,  protein  hydrolysis,  29, 
54,  55 \  carbohydrate  group  in  the  pro- 
teins, 33,  182;  digestion  products.  54, 
55;  histone,  62;  elastin,  75;  monanimo 
acids,  83,  84,  85,  88.  89.  91,  101;  in  tlie 
urine,  614,  675;  cystine  and  cvstinuria, 
92,  631,  675;  histidine,  99,  208;  dia- 
mino  trioxydodecanic  acid.  101;  pro- 
teoses in  the  blood,  183;  blood  serum, 
188;  globin,  208;  blood  corpuscles.  219; 
blood  analyses,  238,  239;  quantity  of 
haemoglobin,  239.  243:  iron  prepara- 
tions, 245;  blood  and  air  dilution.  246; 
adrenalin,  278;  sugar  formation,  296; 
cholesterin,  334,  335;  duodenal  secre- 
tion, 377;  tniJsin,  394,  395,  417;  pro- 
tein absorption,  400.  413,  416,  417; 
ovalbumin,  507;  milk,  529,  536;  poly- 
peptides in  urine,  551,  614;  glycylglycin, 
behavior  in  animal  body,  630;  Bence- 
Jones  protein,  645;  alcohol,  755;  dipep- 
tides  from  protein  bodies,  35;  spongin, 
81 

Abel,  J.,  epinephrin,  278;  spermin,  496; 
carbamic  acid,  552;   melanines,  689 

Abeles,  M.,  sugar,  118;  uric  acid,  186; 
liver  sugar,  295;  carbohydrates  in  the 
urine.  608 

Abelmann,  M.,  protein  digestion,  418;  fat 
absorption.  425 

Abelsdorf.  G..  490 

Abelous,  J.,  enzymes,  8,  17-20 

Ach,  L.,  568 

Achard,  Ch.,  185 

Ackermann,  D.,  195 

Adamkiewicz,  A.,  protein  reaction,  42; 
proteoses,  nutritive  value  of,  746 

Aducco,  v.,  acidity  of  urine,  544;  urine 
poison,  615 

Aders,  R.  H.,  hydrolysis  of  gelatine,  78, 
83,  84 

Adler,  O.,  pentose,  112;  levulose,  119; 
blood,  217,  649 

Adler,  R.,  pentose,  112;  levulose,  119; 
blood,    217,    649 

Adrian,  C,  594 

Adriance,  J.,  human  milk,  531 ;  colostrum, 
531 

Adriance,  V.,  human  milk,  531;  colos- 
trum, 531 


Afanassiew,  M.,  333 

Albanese,  M.,  579 

Albert.  R.,  10 

Albertoni,  P..  bile,  308;  trypsinogen,  390; 
sugar,  absorption  of,  419 

Albrecht,  E.,  193 

Albro,  A.,  393 

Albu,  A.,  urine  poisons,  615;  mineral 
metabolism.  616,  625,  719,  735,  737 

v.  Aldor,  L.,  644 

Aldrich.  J.  B.,  adrenalin,  278;  skunk, 
secretion  of,  692 

Alexander.  F.,  proteoses,  55,  522 

Alexandroff.  D.,  102 

V.  Alfthan,  K.,  carbohydrates  of  lu-ine, 
609;    glucuronic  acid  in  the  urine,  667 

Allard,  E.,  672 

Allen,  S.,  359 

Allihn,  F.,  sugar  determination,  290, 
663 

Almagia,  M.,  amino  acids  and  carbohy- 
drate formation,  305;  uric  acid  demoli- 
tion, 574 

Almen,  A.,  sugar  test,  116.  655;  xanthine, 
159;  meat,  476;   foodstuffs,  773 

Aloy,  J.,  enzymes,  S,  19 

Alsberg,  C,  pseudonucleic  acid,  46;  nu- 
cleic acid.  154 

Altmann,  147 

Altmann,  R.,  nucleic  acids,  152,  156 

Ambronn.  H.,  686 

Amermann,  G.,  pepsin  digestion,  358; 
stomachic   digestion,   370 

Amiradzibi.  S.,  457 

Amthor.  K,  fats,  135,  442 

Andersson,  J.,  277 

Andrlik.  K,  89 

V.  Anrep.  B..  carbon  monoxide  haemoglo- 
bin. 207;    benzoic  acid,  588 

Anselm.  R..  326 

Ansiaux,  G.,  protein  coagulation,  40; 
fibrinogen  and  phosphorus  poisoning, 
172 

Anthon.  115 

Araki.  T..  met  haemoglobin.  204;  sulphur- 
met  haemoglobin.  207;  glycosuria,  299; 
nucleic  acids.  395;  lactic  acid,  460,  461- 
463;  in  urine.  608;  acetoacetic  acid, 
672;    chitin,  686;    chitosan,  687 

Ardin-Delteil,  P.,  694 

813 


814 


INDEX  TO  AUTHORS. 


Argutinsky,  P.,  meat,  476,  720;  perspir- 
ation, 694 

Armstrong,  E.  F.,  enzymes,  13,  16; 
osones,  107 

Arnheim,  J.,  303 

Arnold,  J.,  277 

Arnold,  V.,  neutral  hsematin,  208;  aceto- 
acetic  acid,  672 

Arnschink,  L.,  423 

Arnstein,  R.,  581 

Aron,  H.,  colloids,  41;  bone  eartlas,  436, 
438 

Arrhenius,  S.,  190 

Arronet,  H.,  238 

Arteaga,  T.  F.,  298 

Arthus,  M.,  blood  coagulation,  170,  171, 
226,  227,  229,  231;  fibrinolysis,  174; 
glycolysis,  185;  blood  lipase,  185;  peri- 
toneal fluid,  231;  sugar  formation,  295; 
casein,  519;    rennin  coagulation,  520 

Ascherson,  517 

Ascoli,  A.,  pyrimidine  bases,  152,  164; 
nucleic  acids,  152;  plasminic  acid,  156; 
protein  absorption,  414;  placenta,  512; 
urea  formation,  550;  uric  acid  demoli- 
tion, 574 

Asher,  L.,  blood-sugar,  184;  lymph,  250, 
254-256;  saliva,  340;  protein  absorp- 
tion, 414;   lactic-acid  formation,  461 

Aso,  K.,  19 

Astaschewsky,  469 

Athanasiu,  J.,  blood  coagulation,  234; 
fatty  liver,  283;   fat  formation,  443 

Atwater,  W.  O.,  metabolism,  471,  474,  716, 
726,  748;  respiration  apparatus,  713, 
722;  alcohol  in  metabolism,  754;  diets, 
764,  767,  769 

Aubert,  H.,  695 

Austin,  A.  E.,  290 

Austrian,  C.  R.,  571 

Autenrieth,  W.,  oxalic  acid,  583;  potas- 
sium, 623 

Azemar,  L.,  670 

Ayres,  W.  C,  489 

Akermann,  J.,  365 

Baas,  H.,  cleavage  of  esters,  389;  intestinal 
putrefaction,  586;  demolition  of  tyro- 
sine, 633 

Babkin,  B.,  oil  soaps  and  pancreatic 
secretion,  385;  urea  formation,  551 

Babcock,  523 

Bach,  A.,  autoxidation,  7;  peroxides  and 
oxidases,  7,  8,  17;  enzymes,  8,  19 

Baer,  J.,  544 

Baeyer,  A.,  assimilation,  1,  114;  oxi- 
dations, 6;  indol,  402 

Baginsky,  A.,  bile,  327;  bones,  439 

Baglioni,  S.,  240 

Bainbridge,  F.  A.,  lymph  formation,  255; 
lactase,  386 

Baisch,  C,  carbohydrates  in  urine,  608; 
Vienzoylation,  659 

Baker,  J.  L.,  125 

Balch,  A.,  307 


Baldi,  D.,  jecorin,  283,  481 

Baldoni,  A.,  396 

Balean,  H.,  216 

Balke,  P.,  purine  bases,  159,  163;  phos- 
phocarnic  acid,  457;    episarkine,  580 

Bang,  B.,  539 

Bang,  I.,  histones,  62,  269,  271;  nucleic 
acids,  151,  155,  156,  270;  leucocytes, 
221;  lymph  glands,  269,  272:  thymus, 
270-272;  parachymosin,  361,  362; 
proteoses  in  urine,  644 

Banting,  W.,  770 

Barbera,  A.  G.,  lymph,  250,  254,  255;  bile, 
308;   protein  absorption,  414 

Barbieri,  I.,  phenylalanine,  91;  isochole- 
sterin,  337 

Barker,  L.  F.,  675 

Barral,  185 

Barratt,  W.,  695 

Barth,  H.,  583 

Bartoschewitsch,  S.  T.,  589 

Basch,  K.,  casein  formation,  537;  milk, 
538 

Baserin,  O.,  332 

Bashford,  E.,  586 

Bassow,  349 

Bastianeili,  G.,  378 

Battelli,  F.,  enzymes,  20,  21 

Bauer,  605 

Bauer,  J.,  protein  absorption,  412;  fatty 
degeneration,  443 

Bauer,  M.,  100 

Bauer,  R.,  73 

Baumann,  E.,  diamines,  24;  in  urine  and 
faeces,  615,  676;  cystine  and  cystinuria, 
93,  675;  thiolactic  acid,  94;  carbohy- 
drates, benzoylation  of,  117,  659; 
iodothyrin,  276;  diamidation,  305;  pu- 
trefaction and  products  produced,  401, 
586,  588;  hippuric  acid,  586;  ethereal 
sulphuric  acid,  588,  592,  637;  phenol  in 
urine,  590;  pyrocatechin,  591;  hydro- 
quinone,  591;  urine  indican,  592,  593; 
bile  acids,  597;  oxyacids,  597;  homo- 
gentisic  acid,  597-600;  carbohydrates  in 
the  urine,  608;  sulphuric  acid  of  the 
urine,  estimation  of,  622;  sarcosin,  de- 
struction of,  631;  demolition  of  com- 
povmds  containing  sulphur,  632;  be- 
havior of  aromatic  bodies,  633,  636, 
639 ;  mercapturic  acids,  639 ;  benzoyl  cys- 
tine, 676 

Baumann,  K.,  60 

Baumgarten,  O.,  300 

Baumstark,  F.,  brain,  487;  protagon,  481; 
cholesterin,  480;   urinary  pigments,  650 

Bayer,  H.,  57 

Bayliss,  W.  M.,  intestinal  enzymes,  379; 
erepsin,  380,  391;  enterokinase,  379, 
383;  trypsinogen  and  trypsin,  383,  386, 
390,  392;   secretin,  384 

Beatty,  W.  A.,  35 

Beaumont,  W.,  gastric  fistula,  349;  gas- 
tric digestion,  368 

Beccari,  L.,  326 


INDEX  TO  AUTHORS. 


815 


Bechamp,  A.,  crj'stalline  lens,  493;  oval- 
bumin, 507;   milk  dextrin,  525 

Bechhold,  H.,  656 

Beck,  A.,  urobilin,  603;  urine  poisons, 
615 

Beck,  0  ,  471 

Beck,  R.  F.,  538 

Beckmann,  E.,  692 

Beckmann,  W.,  624 

Becquerel,  A.,  blood,  243;    milk,  534 

Beebe,  S.  P.,  166 

Begar,  C,  536 

Behrend,  R.,  568 

Beier,.  K..  330 

Beitler,  C,  102 

Bellamy,  H.,  385 

Belloni,  E.,  523 

Bence,  J.,  224 

Bence-Jones,  H  ,  645 

Bendrix,  E.,  pentoses,  72,  110,  111;  glyco- 
gen formation,  292 

Benedicenti,  A.,  formalin  protein  com- 
bination, 49;  alcohol  in  metabolism, 
754;  light  and  metabolism,  761 

Benedict,  F.  G.,  metabolism  in  work,  471, 
474;  protein  sparing,  748;  alcohol  in 
metabolisin,  754 

Benedict,  H.,  elimination  of  sulphur,  471, 

'H611 

Benedikt,  R.,  fats,  134,  137 

Benrath,  A.,  364 

Berard,  E.,  protein  coagulation,  40; 
ovglobulin,    .506;     ovalbumin,   507 

Berdez,    J.,    melanins,    688 

Berenstein,  M.,  409 

Bergell,  P.,  carbohydrate  groups  in  pro 
teias,  33,  182;  protein  hydrolysis.  35, 
395;  amino  acids,  92;  lecithm,  147; 
adrenalin,  278;  placenta.  512;  glycyl- 
glycine.  behavior  in  the  body,  630; 
.5-oxybutvric  acid,  674 

Berger,  W.  M..  344  ^ 

Bergh,  E.,  elastin,  75 

v.  der  Bergh,  615 

Bergin,  T.  .1.,  407 

V.  Bergmann,  G.,  residual  nitrogen,  183; 
taurine    from   cystine,   331 

Bergmann.  P.,  thyroidea,  276;  pseudo- 
pepsin,  355;    CEPCum,  427 

Bergmann,  W..  620 

Berlatzki,  G.  B.,  401 

Berlioz,  A.,  616 

Berlinerblau.  M.,  241 

Bernard,  Claude,  glycogen  148,  287 
glycolysis,  184;  sugar  of  the  blood,  240 
sugar  formation,  295-298;  diabetes,  299 
pancreas,  382,  389;  fat  splitting.  389 
fat  absorption,  424;  muscles,  glycogen 
consumption  in,  468 

V.  Bernek.  M.,  14 

Bernert,  R.,  oxyprotic  acids,  31;  mon- 
oxvstearic  acid,  131;  pseudochylous, 
262 

Bernheim,  A.,  transudates,  261,  263 

Bernheim,  R.,  623 


Bernstein,  J.,  477 

Bernstein,  N.  O.,  382 

Bert,  Paul,  mammary  gland,  514,  539; 
blood  gases,  697,  706 

Bertagnini,  C,  636 

Berthelot,  M.  P.,  fat  splitting,  389;  calori- 
metric  determinations,  .511,  724 

Bertin-Sans,  H.,  blood  pigments,  204, 
207 

Bertrand,  G.,  oxidation  enzymes,  8,  18, 
19;  arsenic,  166,  275,  685;  adrenalin, 
278;  epidermis  formations,  685;  poi- 
sons, 693 

Bertz.  F.,  440 

Berzelius,  J.  J.,  347 

Besbokaia,  M.,  .385 

■Bethe,  Alb.,  486 

Bial,  M.,  pentoses,  110,  112;  reage  t,667; 
conjugated  glycuronates,  122,  667; 
diastase  in  the  blood,  185,  295,  297; 
lymph,  251;  glycogen  formation,  294 
sugar  formation,  295 

Bialobrzeski,  M.,  212 

Bialocour,  F.,  371 

Biarnes,  G.,  oxidation  enzymes,  8,  18 

V.  Bibra,  E.,  286 

Bidder,  F.,  buccal  mucus,  341;  saliva, 
347:  gastric  juice,  353;  pancreatic  juice, 
387;  biliary  fistula,  406;  fat  absorption, 
424 

Biedermann,  W.,  18 

Biedert,  Ph.,  530 

Biedl.  A.,  241 

Biel,  J.,  mare's  milk,  529;  human  milk, 
530 

Bielfeld,  P.,  285 

Binet.  P.,  426 

Bienstock,  B.,  408 

Biernacki,  E.,  alkalinity  of  blood,  223; 
blood  analysis,  236;  pepsin,  355;  trj'p- 
sin,  390;  putrefaction  processes,  405, 
407, 588 

Bierrj-,  A.,  lactase  formation,  386;  mal- 
tase,  388 

Biffi,  X.,  522 

Bins;.  H.  J.,  jecorin  and  blood  sugar,  184, 
283.  296 

Binz,  C,  631 

Biot,  J.  B..  711 

Biscaro,  G.,  523 

Bischoff,  Th.,  716 

V.  Bitto,  Bela,  671 

Bizio,  G.,  695 

Bizio,  J.,  287 

Bizzozero,  J.,  blood  plates,  222,  227 

Bjerre,  P.,  755 

Blachstein,  A.,  459 

Blankenhorn,  E.,  protagon,  481,  482 

Bleibtreu,  L.,  blood  analyses,  235;  quan- 
tity of  plasma.  2.38;  urea  estimation 
561;  protein  requirement.  765 

Bleibtreu.  M..  blood  analyses.  235;  quan- 
tity of  plasma,  238;  respiratory  quo- 
tient, 446 

Bleile,  A.  M.,  240 


816 


INDEX  TO  AUTHORS. 


Blendermann,  H.,  tyrosine,  behavior  in 
the  body,  305,  633;  oxyhydropara- 
coumaric  acid,  597 

Bliss,  C.  L.  49 

Bhx,  M.  G.,  236 

Bloch,  C,  587 

Blondlot,  N.,  406 

Blum,  F.,  halogen  proteins,  30;  Millon's 
reaction,  4.;;  protogen,  49;  thyroid 
gland,  277;  adrenalin  glycosuria,  279 

Bliun,  L.,  autolysis,  23;  protein  nitrogen, 
27;  cystine,  demohtion  of,  631;  pro- 
teoses, nutritive  value  of,  747 

Bhunenthal,  F.,  acetone,  32,  668;  nucleo- 
proteids,  72,  282;  pentoses,  110,  666; 
glycogen,  292;  assimilation  limit,  420; 
hippuric  acid,  587;    vu'ine  indican,  593 

Boas,  J.,  chymosin,  361;  reagent  for  lactic 
acid,  374;    for  hydrocliloric  acid,  374 

Bocarius,  N.,  496 

Bock,  C,  blood  sugar,  297;   diabetes,  299 

Bock,  J.,  blood  pigments,  206, 207;  haemo- 
globin and  gases,  207 

Badlander,  G.,  754 

Bodon,  K,  260 

Bodong,  A.,  blood  coagulation,  231;  hiru- 
din. 233 

Boedeker,  C,  591 

Boedtker,  E.,  urine  nitrogen,  548;  clilor- 
ine  estimation,  617 

Boehm,  R.,  glycogen,  459,  466 

BoehtHngk.  R.,  731 

Boekelmann,  W.  A.,  674 

Boemer,  A.,  60 

Boeri,  G.,  oxalic  acid,  582;  sulphur  of 
urine,  611 

Boettcher,  496 

Boettger,  116 

Bogdanow-Beresowski,  343 

Bogomoloff,  Th.,  602 

Bohland,  G.  W.,  269 

Bohland,  K.,  urine  nitrogen,  548;  esti-^ 
niation  of  urea,  557,  561,  613;  of 
ammonia.  625;  uric  acid  elimination, 
569;  protein  requirement.  765 

Bohr,  Chr.,  blood  pigments,  196-200,206; 
oxygen  absorption,  199,  200,  704; 
oxygen  tension,  706-708,  711;  blood- 
gases,  696-698,  704;  carbon  dioxide 
tension,  710;  swimming-bladder,  711; 
oxygen  capacity,  711,  712;  lungs, 
metabolism  of,  704,  714;  secretion  of 
gases,  708,  710;  egg,  incubation  of,  510, 
512 

DuBois-Reymond,  E.,  muscle  work,  469; 
smooth  muscles,  477 

DuBois-Reymond,  R.,  225 

Bokorny,  T,,  reserve  protein,  5;  carbohy- 
drate formation,  114 

Boldireff,  W.  N..  intestinal  enzymes,  378, 
379:    gastric  digestion,  398 

Boll.  F.,  489 

Bonanni,  A.,  bile,  326;  conjugated  glu- 
curonates,  638 

Bondi,  J.,  512 


Bondi,  S.,  sericin,  82;  glycocholic  acid, 
312;  taurocholic  acid,  313;  cholic  acid, 
316.  318;    acetoacetic  acid,  672 

Bondzynski,  St.,  oxyprotic  acid,  31;  kop- 
rosterin,  337;  ovalbmnin,  507;  \irinary 
purines,  578;  oxyproteic  acid  in  the 
urine,  612,  613;  uroferric  acid,  614 

Bonnema,  A.,  517 

Borchardt,  L.,  diastatic  enzyme,  295; 
acetone  estimation,  673 

Bordet,  J.,  precipitins,  186;  blood  coagu- 
lation, 227,  231 

Borissow,  law  of  pepsin  action,  358 

Bori.ssow,  P.,  bitter  principles  ,350;  allan- 
toin,  584 

Borkel,  C,  57 

Bornstein,  K.,  work  and  protein  metabo- 
lism, 471;    protein  feeding,  752 

Boruttau,  H.,  459 

Bosshard,  E.,  85         _ 

Bottazzi,  Ph.,  sea  animals,  osmotic  press- 
ure, 189;  erythrocytes,  220;  intestinal 
protein,  379;  heart  muscles,  451; 
smooth  muscles,  478;  placenta,  512; 
tunicin,  686 

Bouchard,  Ch.,  autointoxication,  25,  280; 
glycogen  formation,  291;  poison  of 
\irine,  615 

Boudet.  183 

Boulud,  conjugated  glucoronates,  122, 184; 
pentoses,  184;  virtual  sugar,  184; 
maltose  in  vu'ine,  667 

Bouma,  J.,  urinary  indican,  595;  reaction 
for  bile  pigments,  653;  /?-oxybutyric 
acid,  674 

Bourcet,  P.,  iodine,  187,  243;  arsenic,  187, 
243;    menstrual   blood,   243 

Bourquelot,  E.,  oxidation  enzymes,  8; 
sugar  elimination,  293 

Bourquet,  F.,  191 

Bo  vet,  v.,  30 

Brahm,  C,  638 

Brand.  J.,  bile,  307,  326,  327 

Brandberg,  J.,  646 

Brandenberg,  K.,  alkalinity  of  the  blood, 
223 

Brandl,  J.,  688 

Brat,  H.,  112 

Brauer,  L.,  329 

Braunstein,  A.,  glycolysis,  303;  estima- 
tion of  urea,  561 

Bredig.  G.,  enzymes,  11,  12;  inorganic 
ferments,  14 

Brenziger,  K.,  675 

Brieger,  L.,  ptomaines,  24;  putrefaction 
products,  401;  skatol,  409,  596;  neuri- 
dine,  481,  486,  502;  urinarj'  indican, 
592,  593;  diamines  in  urine,  615; 
cystinuria,  676;  perspiration,  694 

Brinck,  Julia,  415 

Brion,  A.,  630 

Brocard,  M.,  386 

Brodie,  T.  G.,  fibrin  ferment,  176;  dia- 
betes, 298;   pancreatic  casein,  396 

Brook,  F.  W.,  30 


INDEX  TO  AITHORS. 


817 


Brown,  E.  W.,  cholesterin  esters,  183; 
allantoin,  574,  584 

Brown,  H.,  enzyme  action,  14;  isomaltose, 
125;  hydrolysis  of  starch,  128,  346; 
invertase,  379 

Browne,  C.  A.,  (Jr.),  518 

Brubacher,  H.,  bones,  438,  439 

V.  Brucke,  E.,  enzymes,  11;  peptone,  52; 
coagulation  of  blood,  226;  fibrin.  229; 
glj'cogen,  289,  290;  pepsin,  355-357, 
in  urine,  615;  emulsification  of  fat,  398; 
absorption  of  proteins,  412;  carbohy- 
drates in  the  urine,  608 

Brugsch   H.,  733 

Bruhns,  G.,  164 

Brunner,  Th.,  534 

Bruno,  G.,  action  of  steapsin,  389;  action 
of  bile  and  trj-psin,  393,  397 

Bruno,  B.,  616 

Brunton-Blakie,  455 

de  Bruyn-Lobry',  osamines,  107;  varieties 
of  sugar,  108 

Bryant,  A.  P.,  diets,  767,  769 

Buchanan,  A.,  175 

Buchner,  E.,  fermentation.  10,  11;  zy- 
mases, 11,21;  gases  of  the  lymph,  702 

Buchner,  H.,  fermentation,  10,  21 

Budde,  v.,  664 

Bulow,  K..  proteid,  38;  starch,  126,  128 

Bunz.  R.,  487 

Burger,  L.,  429 

Burker,  K.,  227 

Buffalini,  244 

Bugarsky,  St.,  acid  combinations  of  pro- 
teins, 38;  blood  serum,  molecular  con- 
centration of,  189;  degree  of  dissocia- 
tion, 191;  conductivity  blood  analyses, 
236;  urine,  546 

Bulnheim,  G.,  316 

V.  Bvmge.  G.,  blood  servmi,  188;  blood 
corpuscles,  220;  blood  plasma,  238; 
blood  analyses,  237;  blood  and  air 
dilution,  246;  iron,  of  the  liver,  285, 
534;  absorption,  245;  secretion  of 
hydrocliloric  acid,  364;  gastric  juice, 
371;  cartilage,  434;  haematogen.  503, 
510;  milk.  527.  532.  5.35,  537;  and 
growth,  535  536;  hippuric  acid,  586, 
587;  mineral  requirements,  736;  arti- 
ficial feeding,  739 

Bunsen,  R.,  561 

Buntzen,  J.,  blood  in  starvation,  244; 
blood  and  digestion,  244 

Buraczewski,  J.,  215 

Burchard,  H.,  336 

Burckhardt,  A.  E.    188 

Burian,  R.,  xanthine  oxidase.  18.  158.  273, 
571;  purine  bases,  15S.  163.  454,  470; 
in  the  urine,  579;  formation  of  uric  acid. 
275,  569,  570,  573;  destruction  of  uric 
acid,  574;    histones  in  the  urine,  647 

Burow,  R.,  532 

Busch,  P.  W.,  lymph  formation,  254.  255 

Busch.  W.,  368 

Butlerow,  A.,  113 


Cade,  A.,  352 

Cahn,  A.,  secretion  of  hydrochloric  acid, 
364;  digestion  products,  370;  retina, 
489 

Camerer,  W.,  milk,  526,  527,  531, 533-535; 
urinary  nitrogen,  548;  elimination  of 
uric  acid,  569;  purine  bases,  581;  meta- 
bolism, 756,  577 

Camerer,  W.,  Jr.,  milk,  533,  535;  elimina- 
tion of  ammonia,  624;  perspiration,  693 

Cammidge,  P.  J.,  676 

Campani,  A.,  621 

Campbell,  G.,  ovovitellin,  503;  ovomucin, 
506;    ovalbiunins,  507 

Campbell,  J.  F.,  bilicyanin,  322,  324 

Camps,  R.,  600 

Camus,  L.,  enterokinase,  383;  secretin, 
484;    vesiculase,  496 

Cannon,  W.  B..  gastric  movements,  366; 
food,    digestibility,    369 

Cappelli,  J.,  erythrocytes,  220;  smooth 
muscles,  278 

Capranica,  St.,  guanine,  161;  perspira- 
tion, 694 

Carlier,  E.  W.,  blood  coagulation,  226; 
chyle.  251 

Carl  Theodore  (Duke  of  Bavaria).  761 

Carnot.  Ad.,  bone  earth.  436;   dentin,  440 

Carvallo.  J.,  blood  coagulation,  234;  ex- 
tirpation of  the  stomach,  369,  371 

Casali,  A.,  692 

Caspari,  W.,  high  altitudes,  246,  761;  pro- 
tein metabolism,  471,  750;  milk  fat, 
538;   vegetable  diet,  750 

Castaro,  X.,  arginine,  97;  hemicellulose, 
130 

Cathcart,  E.  P.,  glycogen  formation,  294; 
absorption,  415,  417 

Cavazzani,  E.,  cerebrospinal  fluid.  264; 
destruction  of  glycogen,  296;  carbohy- 
drate absorption,  421;  phosphocarnic 
acid,  457;  muscle  work,  468;  semen,  495 

Chabbas,  J.,  491 

Chabrie.  C,  440 

Chandelon,  Th.,  468 

Chaniewski.  445 

Chanoz.  M..  284 

Charcot,  J.  M.,  496 

Chassevant,  A.,  574 

Chauveau,  A.,  sugar  formation,  306;  fat 
formation,  444-446;  muscle  work,  468, 
473 

Chigin.  P.,  350 

Chittenden,  R.  H.,  proteoses  and  peptones, 
51,  52,  56,  60;  keratin,  73;  elastin,  75, 
76;  gelatine.  77,  79;  saliva.  343-346; 
peptic  digestion,  358,  359;  gastric  diges- 
tion, 369:  trj^Jtic  digestion.  393; 
tendon  mucoid.  428.  435;  mvosin,  450; 
neurokeratin,  73,  480.  488,  489;  nutri- 
tive requirements,  766 

Chodat,  R..  peroxides  and  peroxidases, 
7,8,  17,  19;   enzymes.  8,  19 

Chossat,  Th.,  metabolism  in  starvation, 
828,  732 


818 


INDEX  TO  AUTHORS. 


Christenn,  G.,  531 

Cliristensen,  A.,  646 

Ciamician,  G.,  403 

Cingolani,  M.,  568 

Citron,  H.,  623 

Clar,  C,  569 

Claus,  R.,  303 

ClayiDton,  J.  L.,  23 

Clemens,  Paul,  conjugation  of  glucuronic 
acid,  638;    Ehrlich's  urine  test,  675 

Clemm,  C.  G.,  533 

Clerc,  A.,  185 

Cleve,  P.  T.,  316 

Cloetta,  M.,  hsematin,  210,  212 

Cloez,  330 

Clopatt,  A.,  alcohol  and  metabolism,  754 

Closson,  O.  E.,  8 

Cohn,  Felix,  371 

Cohn,  M.,  saliva,  343;  pancreas  secretion, 
387 

Cohn,  R.,  protein  hydrolysis,  29;  leucine, 
85;  leucinimide,  87;  tyrosine,  89,  633; 
amino  acids  and  carbohydrate  forma- 
tion, 306;  amino  benzoic  acids,  636; 
nitrobenzaldehyde,  636;  furfurol,  637; 
pyridine,  639 

Cohn,  Th.,  blood,  189;  sperm  crystals,  496; 
allantoin,  584 

Cohnheim,  J.,  ptyalin,  344;  urine  dias- 
tase, 616 

Colmheim,  O.,  alcoholic  fermentation,  21; 
proteins,  26,  38;  glycolysis,  302,  303; 
erepsin,  378,  380,  416;  pancreatic  juice, 
384;    apparent  feeding,  745 

Cohnstein,  J.,  243 

Cohnstein,  W.,  alkalinity  of  the  blood, 
223;    lymph  formation,  256 

Colasanti,  G.,  metabolism  of  muscles,  467; 
lactic  acid,  469,  608;  xanthocreatinine, 
567 

Cole,  S.  W.,  protein  reaction,  42,  43;  tryp- 
tophane,   102,    103 

Colenbrander,  M.,  glycolysis,  185 

Collmann,  695 

Colls,  P.  C,  61 

Comaille,  A.,  lactoprotein,  523;   milk,  537 

Connstein,  W.,  185 

Conradi,  H.,  autolysis,  23,  281;  anti- 
thrombin,  234;    intestinal  bacteria,  408 

Constantinidi,  A.,  foodstuffs,  421;  vege- 
table diet,  750 

Contejean,  Ch.,  blood  coagulation,  234; 
phlorhizin  diabetes,  298;  gastric  juice, 
353;  acid  secretion,  364;  pyloric  secre- 
tion, 365 

Copemann,  M.,  649 

Coranda,  G.,  urea  formation,  551;  am- 
monia elimination,  624 

Cordua,  H.,  216 

Corin,  G.,  protein  coagulation,  40;  fibrin- 
ogen, 172;  ovoglobulin,  506;  ovalbu- 
min, 507 

Coronedi,  G.,  442 

Corvisart,  L.,  390 

Le  Count,  E.  R.,  101 


Courant,  G.,  milk,  515,  520,  529;  casein 
salts,  519 

Cousin,  H.,  504 

Couvreur,  E.,  291 

Cramer,  C.  D.,  fibrinogen,  173;  blood  co- 
agulation, 229 

Cramer,  E.,  fibroin,  82;  sericin,  82;  per- 
spiration, 694 

Cramer,  Tr.,  418 

Cramer,  W.,  protagon,  481 ;  hippuric  acid, 
586; 

Cremer,  M.,  pentoses.  111,  290;  glycoly- 
sis, 185;  glycogen,  288-291,  293; 
phlorhizin  diabetes,  298;  sugar  from 
glycerine,  308;    fat  formation,  445 

Croftan,  A.,  bile  acids  in  blood,  238; 
suprerenal   capsule,   279 

Croft-Hill,  A.,  16 

Croner,  W.,  375 

Cronheim,  W.,  375 

Croockewitt,  J.  H.,  82 

Cummins,  G.  W.,  trypsin  digestion,  393; 
myosin,  450 

Cunningham,  R.  H.,  425 

Curtius,  Th.,  peptide  syntheses,  34;  cholic 
acid,  315  . 

Cutter,  W.  D.,  tendon-mucin,  66,  428; 
saliva,  340 

CybuLski,  N.,  278 

Cerny,  T.,    lactic  acid  fermentation,  461 

Czerny,  A.,  221 

Czerny,  F.,  alcoholic  fermentation,  21 

Czerny,  V.,  extirpation  of  stomach,  369; 
absorption,  413 

Czerny,  Zd.,  644 

Daddi,  L.,  fat  in  blood,  244;  milk  fat,  538 

Daenhardt,  C,  702 

Dakin,  H.  D.,  arginase,  36,  97,  550;  pro- 
tamines, 63,  64,  84,  95,  97,  101;  hexone 
bases,  98,  100;  adrenalin  substances, 
278;     ester    cleavage,  284 

Daland,  J.,  237 

Danilewsky,  A.,  protein  sulphur,  28; 
plasteines,  56,  363;  anti  enzymes,  372, 
379;  muscle  protein,  448,  451,  475; 
milk  globules,  517 

Danilewsky,  B.,  724 

Danilewsky,  W.,  146 

Dareste,  C,  testicles,  495;  yolk  of  egg,  502 

Darmstadter,  E.,  674 

Darmstadter,  J.,   692 

Darmstadter,  L.,  337 

Dastre,  A.,  fibrinogen,  172;  fibrinolysis, 
174,  175;  blood  coagulation,  227,  234; 
glycogen,  222,  251,  295,  296;  liver,  282, 
286;  sugar  elimination,  293;  bile,  307, 
324,325;  in  stomach,  398;  trypsinogen, 
386;    fat  absorption,  424 

Dautzenberg,  P.  J.  W.,  636 

Dauwe,  F.,  12 

Davis.  W.  S.,  689 

Day,  H.,  367 

Dean,  A.  L.,  inulin,  127;  protein  regener- 
ation, 417 


INDEX  TO  AUTHORS. 


.  819 


Decaisne,  E.,  536 

Dehn,  W.  M.,  618 

Delezenne,  C,  blood  coagixlation,  170,  226, 
231,  234,  255;  intestinal  juice,  378; 
enterokinase  and  pancreatic  juice,  379, 
383,  386;  secretin,  384 

Delfino,  A.,  512 

Demant,  B.,  378 

Deniges,  G.,  tyrosine,  91;  uric  acid,  576; 
determination  of  purine  bases,  582; 
homogentisic  acid,  600 

Denis,  P.  S.,  177 

Derrien,  E.,  methaemoiglobin,  202,  205 

Desgrez,  A.,  291 

DeucherJ  P.,  pancreas,  418,  425 

Devoto,  L.,  determination  of  proteoses, 
60,  61,  644 

Devillard,  P.,  264 

Diaconow,  C,  lecithin,  143,  147 

Diamare,  V.,  pancreas,  302,  381 

Diels,  O.,  cliolesterin,  334,  335 

Diesselhorst,  G.,  693 

Dietschy,  R.,  644 

Dietze,  A.,  393 

Dillner,  H.,  506 

•  Disque,  L.,  urobilin  and  urobilinogen,  601, 
603,  604 

Ditthorn,  Fr.,  galactosamine,  65,  71,  121 

Dittrich,  P.,  205 

Dock,  F.  W.,  299 

Dorpinghaus,  Th.,  carbohydrate  groups  in 
proteins,  33,  182;  products  of  protein 
hydrolysis,  74,  84,  85,  88 

Dombrowski,  St.,  oxy|Droteic  acids  in 
urine,  613;  viroferric  acid,  614;  urinary 
ptomaines,  615 

de  Dominicis,  N.,  300 

Donath,  J.,  264 

Donne,  A.,  651 

Dormeyer,  C,  137 

Doyon,  M.,  fibrinogen,  172;  lipases,  185, 
284;  bile,  308,  309,  329;  biliverdin, 
324 

Dragendorff,  D.,  652 

Drechsel,  E.,  oxidation,  8;  protein  sub- 
stances, 26-29,  36,  38;  gorgonin,  82; 
diaminoacetic  acid,  97;  hexone  bases, 
78,  98,  100;  lysuric  acid,  9S;  purine 
bases,  159;  thyroidea,  276;  jecorin, 
283;  urea  formation,  550,  552;  car- 
bamates, 552;  silicic  acid  ester,  685 

Droop-Richmond,  IL,  517 

Dubelier,  D.,  753 

Dvicceschi,  V.,  blood  coagulation,  235; 
heart-muscle,  451 

Duclaux,  E.,  protein  coagulation,  40; 
milk  fat,  518 

Dull,  G.,  128,  129 

During,  Fr.,  437 

Dufourt,  L.,  bile,  308,  309,  329;  work, 
carbohydrate  consumption  in^  468 

Duggan,  C.  W.,  40 

Dumas,  J.  A.,  553 

Dunlop,  J.  C,  work  and  protein  meta- 
bolism, 471;  oxalic  acid,  582 


Ebstein,  E.,  pentoses,  72,  110,  111 

Ebstein,  W.,  saliva,  345;  pyrocatechin, 
591;  urinary  calculi,  680;  diet  cures, 
770,  771 

Eckhard,  C,  340 

Edkins,  J.  S.,  pepsinogen,  364;  pancreatic 
rennin,  396 

ver  Eecke,  A.,  756 

Ehreiifeld,  R.,  protein  hydrolysis,  29; 
oxyglutaric  acid,  32;  halogen  proteins, 
32;  leucine,  86;  tyrosine,  90 

Ehrenreich,  M.,  391 

Ehrenthal,  W.,  409 

Ehrlich,  E.,  719 

Ehrlich,  F.,  87 

Ehrlich,  Paul,  aldehyde  reaction,  121,  675; 
precipitines,  186;  bilirubin  reaction, 
321,  654;   urine  tests,  674 

Ehrstrom,  R.,  histone,  62;  elimination  of 
phosphorus,  620;   peptonuria,  643 

Eichholz,  A.,  globulins,  180;  glycoproteids, 
506;   urobilins,  602 

Eichhorst,  XL,  413 

Einhorn,  M.,  403 

Eiselt,  Th.,  651 

Ekbom,  A.,  317 

Ekehorn,  G.,  619 

van  Ekenstein,  Alberda,  108 

Ekholm,  K,  768 

Ekunina,  M.,  461 

Embden,  G.,  cysteine  and  cystine,  28,  92, 
94;  proteoses  in  the  blood,  183,  415; 
ethereal  sulphuric  acids,  280;  conjugated 
glucuronic  acids,  280;  glycolysis,  303; 
amino  acids,  carbohydrate  formation, 
305;  passage  of,  into  urine,  614;  pro- 
tein regeneration,  416;  lactic-acid  for- 
mation, 416;   acetone  formation,  262 

Embden,  H.,  598 

Emerson,  R.  L.,  394 

Emich,  Fr.,  406 

Emmerling,  A.,  74 

Emmerling,  O.,  enzymes,  12,  16,  17;  re- 
version of  sugar;  125 

Ellenberger,  W.,  gastric  digestion,  370; 
protein  absorption,  413;  milk,  529; 
appendix,  397 

Ellinger,  A.,  isoserine,  96;  diamines,  97, 
98;  diamine  acids,  97,  98;  indol-acetic 
acid  and  indol-propionic  acid,  102;  co- 
agulation of  bood,  234;  lymph  forma- 
tion, 255,256;  pancreatic  secretion,  387; 
urinary  indican,  592-594;  kynurenic 
acid,600;  proteoses, nutritive  valueof,746 

Elvove,  E.,  8 

Ely,  J.,  saliva,  343,  345 

Engel,  H.,  lipases,  363,  389 

Engelmann,  G.  J.,  471 

Engler,  C,  6,  7 

Eppinger,  258 

Erb,  W.,  38 

Erben,  Fr.,  oxystearic  acid,  131;  blood 
in  disease,  246,  247;  fat  of  chyle,  251; 
nitrogen  partition  in  the  urine,  549; 
urein,  563 


■820 


INDEX  TO  AUTHORS. 


Erlandsen,  A.  W.  E.,  143,  144 

Erlanger,  J.,  427 

Erlenhieyer,  E.,  leucine,  85;  tyrosine, 
89,  90 

Erlenmeyer,  E.  jr.,  tyrosine,  89;  cystine, 
93;   serine,  96 

Esbach,  G.,  645 

Estor,  A.,  711 

Etard,  A.,  457 

Etti,  C,  512 

Eulenburg,  A.,  295 

V.   Euler,   H.,   catalysis,    14,    15,   20 

Eves,   F.,   345 

Ewald,  Aug.,  keratin,  73;  gelatine,  78; 
hsematoidin,  216;  digestion  of  gelatine, 
395;  of  cartilage,  395;  visual  purple, 
489;   corpora  lutea,  498 

Ewald,  C.  A.,  protein  absorption,  413; 
gases  of  transudates,  703;  metabolism 
and    light,    761 

Ewan,  Th.,  6 

Eykman,  C,  isotony,  193;  blood-cor- 
puscles, 195;  blood  analysis,  236;  inhab- 
itants   of    tropics,    762 

Eymonnet,  614 

Fabian,  E.,  294 

Falck,  F.  A.,  744 

Falck,  C.  Ph.,  744 

Falk,  Ernst,  758 

Falta,  W.,  alcaptonuria,  598;  protein 
metabolism,  719,  741;  artificial  nutri- 
tion, 738 

Falloise,  A.,  secretion  of  bile,  309;  intes- 
tinal enzymes,  379;  carbon  dioxide  ten- 
sion, 710;  metabolism,  761;  lipase,  363 

Fano,  G.,  171 

Farkas,  G.,  191 

Farkas,  K.,  512 

Farnsteiner,  K.,  136 

Farwick,  B.,  475 

Faust,  E.,  sepsine,  24;  gelatine,  77; 
samandarin,  692 

Favre,  P.  A.,  694 

Fawitzki,  A.,  554 

Fedeli.  C,  590 

Feder,  L.,  551 

Fehling,  H.,  reagent,  116;  sugar  estima- 
tion, 660 

Fehrsen,  A.,  244 

Feinschmidt,  J.,  303 

V.  Fenyvessv,  Bela,  conjugated  glucuronic 
acids,  610,  638 

Fermi,  CI.,  fibrin,  175;  foodstuffs,  digesti- 
bility of,  368;  autodigestion  of  the 
stomach,  372;    trypsin  test,  392 

Fernet,  E.,  701 

Filehne,  W.,  32.9 

Filhol,  539 

Fingerling,  G.,^36 

Finkler,  D.,  oxidations,  3,  723 

Fischer,  Emil,  enzyme's,  13,  16;  protein 
hydrolyses,  29,  35.  54,  55,  74,  78,  82; 
peptides,  35,  55,  395;  amino  acids,  83- 
92,  101;  ornithine,  97;  diamhiotri- 
oxydodecanoic  acid,    101;    oxypyrroli- 


dincarboxylic    acid  ,      102;       carbohy- 
drates, 105-109,  113,  114,  120;   glucosi- 
aes,    108;     sugar  syntheses,    114;    glu- 
cosamine,   107     108,    120;     glucuronic 
acid,     121;      isomaltose.,     125;      purine 
bases,   156,   157,    160-162;    pyrimidine 
bases,  164,  16;5;    trypsm,  394,  395,  416; 
lactose   fern'^entaition,    524;     uric   acid, 
568;   urinary  purines,  579;    conjugated 
glucuronic  acids,  610 
Fischer,  Chr.,  83 
Fischler.  M.,  324 
Flatow,  R.,  578 
Flaum,  M.,  3o8 
Fleckseder,  R.,  343 
Fleig,  C,  bile,  309;   pancreatic  juice,  384; 

sapokrinm,  385 
Fleischl,  E.,  hsemometer,  219;    formation 

of  bile,  330 
Fleitmann,  28 
Fleroff,  A.,  63 
Fletcher,  H,  M.,  347 

Flint,  A.,  stercorin,  337;    work  and  pro- 
tein catabolism,   471 
Florence,  496 
Floresco,    N.,    blood    coagulation,    234; 

liver,  282,  286;   bXliprasiai,  324 
Flueckiger,  M.,  urine,  609 
Foa,  C,  saliva,  343;   milk,  515,  530 
Folin,  O.,   animal  gum,  67;    alkalinity  of 
blocd,  223;    muscle  rigor,  466;    acidity 
of  urine,  545-;    urea,   561,   562;    urein, 
563;     creatinine,    563,    565,    567,    744; 
uric  acid,  569,  571,  578,  744;    sulphur 
of  urine,  611,  744;    sulphuric  acids  of 
urine,    623;     ammonia,    625;     protein 
metabolism,  744 
Fordos,  M.,  269 
Forrest,  J.  R.,  437 
Forssner,  G.,  614 

Forster,  J.,    transfusion    of    blood,    248; 
mineral   metabolism,    735;    water    and 
metabolism,  753;     dietaries,   764,  770, 
771 
Fraenckel,    P.,    blood,    determination   of 
alkalinity,     192;      methods     oi     blood 
analysis,  236;    gastric  juice,  354 
Fraenkel,  A.,  urine  in  phosphorus  poison- 
ing,  554;    actioin  of  diluted  air,   705; 
dyspnoea  metabolism,   758 
Fraenkel,  Sig.,  proteins,  28,  33,  45;   pep- 
tones, 53,  57,  60;   thiolactic  acid,  28,  94; 
histidine,  99;    thyreoidea,  276;    forma- 
tion of  glycogen,   294;     gastric    juice, 
363;     homogentisic   acid,   599;    chitin, 
686 
Framm,  F.,  glutin,  79;    sugar  and  alkali, 

115;    glycolysis,   185 
Franchimont.  A.  P.,  686 
Franks  O.,   estimation  of  fat,    137;     fat 
absorption,  421-423;  cui'are  poisoning, 
467 
Frankenstein,  W.,  6 
Frankland,  E.,  724 
Franz,  Fr.,  171 
Fredericq,    L.,    protein    coagulation,    40; 


INDEX  TO  AUTHORS. 


82 1 


serglobiilin,  ISO;  osmotic  pressure,  190; 
hsemocyanin,  219;    blood  coagulation, 
226;  blood  gases,  698,  706,  710 
Fremy,  E.,  muscles  of  cephalopods,  478; 

yolk-splierules,  509 
Frentzel,   J.,    glycogen,    288,  290;    work 
and  fat  catabolism,  472,  474;   nitrogen 
in  meat  and  meat  extracts,  477;  calories 
and  nitrogen,  727 
Frericlis.   F.   Th.,   synovia,   266;     human 
bile,   326;    saliva,   347;    uric  acid,  de- 
struction of,  573 
Freudberg,  A.,  223 

Freund,  E.,  serglobulin,  179;  h;T'matin- 
ogen,  216;  coagulation  of  blood,  226; 
determination  of  chlorine,  617;  star- 
vation metalx)lism,  733;  cellulose  in 
consumptives,  714 
Freund,  O.,  733 
Frevtag.  Fr.,  cerebrosides,  268,  482,  483, 

484;   protagon,  481-483 
Friedenthal,   H.,   oxidases,   15;    determi- 
nation of  alkalinity,  192;    pepsin,  355; 
digestion    oi    starches,    367;     protein 
assimilation,  412 
Friedjung,  J.  K.,533 
Friedlander,  G.,  413 

Friedmann,  E.,  sulphur  of  proteins,  28; 
proteoses,   56;    thiolactic  acid,  74,  94; 
thioglycolic  acid,  74;    cystin,  93;   taur- 
ine, 95,  321 ;    adrenalin,  278;    mercap- 
turic  acids.  639 
Friend,  W.  M.,  erythrocytes,  194;    peri- 
cardial fluid,  261 
Fromm,  E.,  638 
Fromme-,  A.,  363 
Frommer,  V.^  671 

Frouin,  A.,  gastric  juice,  351;    intestinal 
juice,    378;     enterokinase,    383;     pan- 
creatic juice,  383 
Fubini,    S.,     respiration    by    skin,     695; 

metabolism  and  light,  761 
Furbringer,  P.,  oxalic  acid,  582;    protein 

reagent,  643 
V.  Fiirth,  O.,  tyrosinase,  18;   peroxyprotic 
acid,   31;    xanthomelanin,   32;    supra- 
renin,  278;    bile  and  fat  cleavage,  398; 
muscle  proteins,  448,  451-453,  477,  478; 
muscle  rigor,  466;    smooth  muscle'^,  477, 
478;    chitosan,     687;     melanins,    689, 
690 
Fuld,   E.,   fibrin   formation,    176;    blood 
coagulation,  231,  232,  234;    chjinosin, 
361,  362;    remiet  coagulation,  520,  521 
V.  Fvinke,  471 

Gabriel,    S,    cystine,    93;     bones,    438; 

teeth,  440;  ovalbumin,  507;  asparagin, 

nutritive  value  of,  747 
Gachet,  J.,  3S'> 
Gad.  J.,  emulsification  of  fat,  398;    mve- 

line,  481 
Gartner,  G.,  219 
Gaglio,  G.,  lactic  acid,  241,  460;    oxalic 

acid,  629 
Galdi,  F.,  258 


Galeotti,  G.,  39 

Galimard,  J.,  74 

Gallois,  459 

Gamgee,   A.,  nueleoproteids,  72;   nucleic 

acids,    153;    oxy haemoglobin,  200,  202; 

int-estinal   juice,    377;     protagon,    481, 

482;  pseudocerebrin,  485 
Ganassini,  D.,  343 
Gamier,  L.,  598 
Garrod,   A.    E.,    htematoporphyrin,   213- 

215,  650;    homogentisic  acid,  598-600; 

vu-ocln-ome,  601 ;  lu-obilin,  603,  605,  606; 

uroerj-tlirin,  607;  cystinuria,    676;  di- 
amines, 676 
Gatin-Gruzewska,    Z.,    seralbumin,    181; 

glvcogen.  287,  288 
Gauhe.  T.,  694 
Gaud.  F.,  115 
Gaule,  J.,  245 


tlnnnus,  271;  glycogen,  290;  fat  forma- 
tion, 444,  445;  muscles,  457;  proteid  of 
egg    white,   507,    508;    xanthocreatine, 

457,   567;   epidermis  formations,  685 
V.  Gebhard,  F.,  467 
Geelmuyden,  H.  C,  acetone  bodies,  669, 

670;  estimation  of,  674 
Geissler,  643 

Gengou,  O.,  blood  coagulation,  227,  261 
V.  Genser,  534 

Gentzen,  M.,  trj-ptopliane,  103;  indol,  593 
Geoghegan,    E.    G.,    cerebrin,    483,    484; 

brain,  mineral  constituents  of.  489 
Geppert,  J.,  alkahnity  of  the  blood,  223; 

oxj'gen  of  blood  and  air  pressure,  705; 

respiration    investigations,     713,     722, 

733;    alcohol,  nutritive. value  of,  754 
Gerard,   E.,   reductases,   19;     cholesterin, 

335;   uric  acid  fermentation,  568 
Gerber,  N.,  531 
Gerhardt,  C,  672 
Gerhardt,  D.,  604 
Gerngross,  O.,  165 
Gertner,  W.,  309 
Gessard,  C,  690 
Geyer,  J.,  658 
Gej'ger,  A.,  598 
Giacosa,  P.,  mucin  substances,  66.  509; 

estimation    of    blood    ])igments,    217; 

frog's    eggs,    509;     urinary    iron,    626; 

demolition  of  aromatic  substances,  634 
Giertz.  H.,  47 
Gies,  W.  J.,  mucin  substances.  66.  67.  68, 

360,  428.  429,  43^;    elastin.  75.  76,  100; 

gelatine,   77,   100;     liexone  bases.   100; 

action  of  ions.  168.  169;    hnnnh  forma- 
tion. 2.i4,  255;    pancreatic  juice.  387; 

Achilles     tendon,      429;       ligamentura 

nucha\  428,  429;  bones,  435;  protagon, 

4S2;    ureine,  563 
Gilbert.  455 

Gilson.  E.,  lecithin,  143,  147;    chitln,  686 
Ginsberg,  S.,  421 
Githens,  Th.  St.,  188 


822 


INDEX   TO  AUTHORS. 


Gittelmacher-Wilenko,  G.,  urine  purines, 

582;   urine  reducing  power  of,  609 
Giunti,  L.,  630 
Gizelt,  A.,  385 
Glaessner,    K.,   ethereal   sulphiuic   acids, 

280;    gastric  enzymes,  355,  362;    Brun- 

ner's  glands,  377;  pancreatic  juice,  387; 

protein  absorption,  413,  416;  kvnurenic 

acid,  600 
Glassmann,  B.,  561 
Gleiss,  W.,  469 
Gley,  E.,    iodine,    187;     coagulation    of 

blood,   234,  235;    lymphogogues,   255; 

enterokinase,  383,  secretin,  484;  vesic- 

ulase,  496 
Glikin,  W.,  137 
Gluzinski,  A.,  369 
Gmelin,  L.,  test  for  bile  pigments,  321, 

322,  652;   saliva,  347 
Gmelin.  W.,  353 
Gogitidse,  S.,  538 
Goldmann,  E.,  cystine  and  cystinuria,  93, 

675,  676;  iodothyrine,  276;   demolition 

of  sulphurized  compounds,  632;    dia- 
mines, 675 
Goldmann,  H.,  215 
Goldschmidt,  C,  556 
Goldsclunidt,  F.,  55 
Goldscluuidt,  H.,  344 
Gonnermann,    M.,    tyrosinases,    18,    90; 

givcocoll,  83;    action  of  trypsin,  395 
Goodbody,  W.,  401 
Goodwin,  R.,  52 
Gorodecki,  H.,  332 
V.  Gorup-Besanez,  E.  F.,  pericardial  fluid, 

260;   human  bile,  326 
Gossip,  B.,  460 
Gossmami,  H.,  pancreas,  382 
Goto,  M.,  protamines,  63,  64;    uric  acid, 

576 
Gottleib,  R.,  urea  in  blood,  240;    iron  in 

the  bile,  326;  in  the  urine,  626;   urinary 

purines,  578;    oxyproteic  acid,  612 
Gourlay,  F.,  275 
de  Graaff,  C.  J.  H.,  645 
Graebe.  C,  412 
Graffenberg-er,  L.,  438 
Graham,  Th.,  39 
Grawitz,  E.,  246 
Green,  E.  H.,  81 
Green,  J.  R.,     fibrinolysis,      174;      blood 

coagulation,  229 
Gregor,  A.,  iJ67 
Gregor,  G.,  623 
Grehant,    N.,    urea    in    blood,    240,    242; 

urea  formation,  553;   fatty  acids  in  the 

urine,  629 
Griffiths,  A.  B.,  neurokeratin,  489;    urine 

poison,  615 
Grimaux,  E.,  protein  synthesis,  34,  234 
Grimbert,  L.,  606 
Grimm,  F.,  604 
Grimmer,  W.,  367 
Grindley,  H.  S..  767 
Griswold,  W.,  345 


Grober,  J.,  355 

Groeber,  A.,  595 

Grohe,  B.,  870 

Groll,  S.,  244 

Grosjean,  A.,  234 

Gross,  A.,  pseudochylous  effusions,  262; 
ovovitellin^  503,  504 

Gross,  O.,  625 

Grosser,  P.,  596 

Grouven,  H.,  475 

Gruber,  D.,  dextrins,  128,  129 

Gruber,  I\I.,  nitrogen  deficit,  718;  water 
and  metabolism,  754 

Griibler,  G.,  38 

Griinbaum,  D.,  512 

Griindler,  J.,  631 

Griinhagen,  A.,  264 

Griiixs,  G.,  195 

Griitzner,  B.,  642 

Griitzner,  P.,  estimation  of  pepsin,  357; 
gastric  contents,  366;  Brunner's  glands, 
377;    pancreatic  diastase,  388 

Grund,  G.,  pentoses,  72,  110,  111 

Grutterink,  645 

Gscheidlen,  R.,  sulphocyanides,  343,  611; 
lactic  acid,  460;    urea  formation,  553 

Gubler,  A.,  lymph,  252;  witch's  milk,  534 

Gumlxjl,  Th.,  27 

Giirber,  A.,  .seralbumin,  181,  182;  serum, 
mineral  constituents  of,  187;  alkalinity  of 
blood,  224;  bile, 329;  amniotic  fluid,  512 

Guerin,  G.,  615 

Giinzburg,  A.,  374 

Guillemonat,  A.,  spleen,  274;  liver,  285 

Guinochet,  E.,  262 

Gulewitsch,  W.,  arginine,  96;  choline,  145; 
nucleic  acid,  154;  thymin,  165;  tryp- 
sin, 395;  extractives  of  the  muscles,  457 

Gullbring,  A.,  314 

Gumilewsky,  378 

Gumlich,  E.,  protein  absorption,  413; 
urinary  nitrogen,  548,  554;  phosphorus 
metabolism,  619 

Gunning,  J.  W.,  670 

Gusserow,  A.,  583 

Guth,  F.,  132 

Gyergyai,  A.,  proteoses,  414,  746 

Haas,  K,  214 

Habermann,  J.,  proteins,  29,  32;    amino- 

acids,  87,  88,  91 
Handel,  M.,  434 
Hansel,  E.  W.,  372 
Haser,  627 
Hiuisermann,  E.,  245 
Hafner,  A.,  132 
Hagermann,  O.,  cutaneous  respiration  of 

the  horse,  695;    work  and  metabolism, 

760 
Hagen,  W.,  164 
Hahn,  M.,  fermentation,  10;    digestion  of 

casein,   521;    Eck's  fistula,   552;    urea 

formation,  552,  553 
Haig,  A.,  569 
Haiser,  F.,  155 


INDEX   TO  AUTHORS. 


823 


Haldane,    J.,    blood    pigments,    204-206; 
estimation  of,  218;    quantity  of  blood, 
248;    oxygen  tension,  707,  708 
Haliff,  E.,  20 

Hall,  W.,  pmine  bases,  163,  408,  454,  5S2; 
absorption  of  iron,  245;   artificial  nutri- 
tion, 739 
Hallauer.  B.,  329 
V.  Hallervorden,  E.,  urea  formation,  551, 

554;  elimination  of  ammonia,  625 
Hallibiu-ton,  W.  D.,  protein  coagulation, 
40,  61;  nucleoproteids,  72;  dextrins, 
129;  cell  globulins.  141,  194;  fibrin 
ferment,  176;  seralbimiin,  181;  blood 
serum,  188;  blood  corpuscles,  194; 
blood  coagulation,  234;  tetroneiAthrin, 
219.  690;  pericardial  fluid.  261;  cere- 
brjspinal  fluid,  264,  488:  liver,  281; 
glycogen,  289;  pancreatic  rennin,  396; 
myxcedema,  429;  bone  marrow,  437; 
muscles,  448-453;  proteins  of  the  brain, 
479;  diseases  of  the  nervous  svstem, 
488;  kidnevs,  542 
Halsey,  J.  T.^  90 
Ham,  C.  E.,  216 

Hamburger,  C,  amyolysis,  185,  345 
Hambm-ger.    H.    J.,    theorv   of   ions,   41; 
colloid^s.    169;     blood  sefmn,   187,   189, 
190;    stroma  of  blood-corpuscles,   192, 
220;    isotony,  193;   permeability  of  the 
blood    corpviscles,    195,    196;     of    the 
muscles,   465;     leucocytes,   220;    alka- 
linity of  blood,   187,  224;    hanph  for- 
mation, 255;    ascitic  fluid,  263;    intes- 
tinal juice,  377,  378;  enterokinase,  383; 
absorption,  427 
Hamburger,  E.  W.,  326 
Hammarsten,  O.,  chymosin,  13;  globulins 
46.179-181;   nucleoalbumin.  47;  mucin 
substances,    66,     67,    68,    509;     helico- 
proteid,  71;    nucleoproteids,     110.  282 
381;      protoplasm,      141;       fibrinogen, 
fibrin  and  blood  coagulation,  173,  176, 
177,  183,  230;   fibrin  globulin,  177,  183; 
seralbimiin,  181,  183:  blood  plasma  and 
serum,    composition   of,    187;     hsema- 
topoq)hyrin,  215,  650;   casein,  179,  520; 
coagulation  with  rennet,   520;    l\Tnph 
gases,  251,  702;    transudates,  258,  260- 
263:    synovial   fluid,   265;     storage  of 
carbohydrate,    294;     bile   mucin,   310; 
bile    acids,    312,    314,    319;     scj^nol 
sulphuric    acids,     310;     dehvdrocholic 
acid.  315;   bile  of  various  anmiais,  307, 
312.  315,  319,  327;  reaction  for  bile  pig- 
ments, 232,  653;    cholohsematin,  324; 
hmnan  bile,   326,   327;     phosphatides, 
325,  328:    urea  in  the  bile,  325,  547; 
saliva,  345;     antichjinosin,  361;     pan- 
creas infusion.  391:    pseudomucin.  500; 
perch-eggs,  504,  509;    lactoprotein.  523; 
urinar\'  protein,  645;   phenylhydrazine 
test,  658 
Hammerbacher,  F.,  347 
Hammerl,  H.,  409 


Hammerschlag,  A.,  222 
Hanriot,    M.,    enzymes,    13;  lipases,   17, 
185;     diabetes,   300;     respiratory   quo- 
tient, 446;  respiration,  713,  722 
Hansen,  C,  lecithin,  143;  protein  regener- 
ation, 417;  fatty  tissues,  441;  yolk-fat, 
504;   milk-fat,  538 
Hansen,  W.,  132 
Hardv,  W.  B.,  169 
Harley,  G.,  600 

Harley,  v.,  hver  and  blood  sugar,  297; 
intestinal  putrefaction,  401;    pancreas, 
418,  425;  large  intestine,  426;  urobilin, 
604 
Harms.  H.,  436 

Harnack,  E.,  ash-free  protein,  38;  iodo- 
spongin,  81;  blood-pigments,  205,  207; 
hydramnion,  513;  oxalic  acid  poisoning, 
593;  m-inary  sulphur.  611;  demolition 
of  chlorine  substituted  methanes,  631; 
gallic  and  tamiic  acid,  637 
Harris,  J.  F.,  protein  nitrogen.  27; 
nucleic  acids,  152,  156;  pyrimidine 
bases,  152,  156 
Hart,  E.,  protein  nitrogen,  27;   proteoses, 

56;   histidine,  99;   hexone  bases,  100 
Hart,  A.  S.,  elastin,  75 
Hartogh,  306 
Hartung,  C,  509 
Hart  well,  J.  A..  52 
Harvey.  W.,  diet  cures,  770,  771 
Hasebroek,  K.,  lecithin,   146,  404;    peri- 
cardial fluid,  261;    digestion  products, 
360 
Haskins,  H.   D.,  vireine,  563;    carbamic 

acid,  563 
Haslam,  H.  C,  salting  out  of  proteins,  39, 

179,  181;   proteoses,  55,  5(3,  60 
Hasselbalch,  K.  A.,  incubation  of  the  egg, 
510;  absorptionof  oxygen,  704;  oxygen 
tension,  711 
Hauff .  534 

Hausmann,  W.,  nitrogen  in  protein  sub- 
stances,    27,     78-      koprosterin,     338; 
cholesterin,  337 
Hawk.    P.    B.,    bones,    435;     water   and 

metabolism,  754 
Hay.  M..  262 

Haycraft.  J.  B.,  protein  coagulation.  40; 
blood  coagulation,  171,  226;  diabetes, 
300;  biliverdin.  323;  test  for  bile-acids, 
652 
Hayem,  G.,  hsematoblasts,  226:  per- 
nicious ansemia.  246:  estimation  of 
hydrochloric  acid.  376 
Hedenius,  J,,  gizzard.  74;    bile  pigment, 

325 
Hedin,  S.  G.,  elastin,  75:  protein  hvdrolv- 
sis.  78:  hexone  bases,  96.  98-100; 
blood-coriniscles,  193-195;  blood  analy- 
sis. 236:  haematocrit.  237;  lienases,  273; 
action  of  trypsin,  392;  enzjnnes  of  the 
muscles,  454 
Hedon,  E.,  pancreatic  diabetes,  301;  ab- 
sorption of  sugar,  419;   of  fat,  425 


824 


INDEX  TO   AUTHORS. 


Heffter,  Aj,  liver.  283;  muscle  reaction  of, 
447,  466;  lactic  acid,  463,  469;  hypo- 
sulphites in  the  urine,  612;  foreign 
bodies  in  the  urine,  629;  synthesis  of 
hippuric  acid,  635 

Heger,  P.,  280 

Heidenhain,  M.,  44 

Heidenhain,  R.,  IjTnph,  250,  253-255; 
traiisudates,  257;  bile  secretion,  307, 
308;  saliva,  341,  437;  stomach,  349, 
350,  363,  365;  pyloric  secretion,  365; 
pancreas  and  its  secretion,  381,  382; 
trypsin,  386,  391;  protein  regeneration, 
416;  sugar  absorption,  420;  smooth 
muscles,  477;  milk,  537 

Heilner,  E.,  754 

Heinemann,  H.  N.,  474 

Heintz,  W.,  fat,  133;  lactic  acid,  460; 
estimation  of  uric  acid,  569,  576 

Heiss,  E.,  439 

Heitzmaim,  C,  439 

Hekma,  E.,  intestinal  juice,  377-379; 
enterokinase,  383;    trypsinogen,  386 

Hele.  T.  Sh.,  599 

Helier,  H.,  609 

Heller,  Fl.,  protein  test,  41,  641;  uro- 
xanthine,  592;  urinary  pigments,  601; 
blood  test,  649;  urinary  calculi,  683, 
684 

Helm,  349 

Helmholtz,  H.,  470 

Helhvig,  smooth  muscles,  477 

Hemmeter,  J.  C,  407 

Henderson,  Y.,  protein  nitrogen,  27; 
protein  regeneration,  416,  417 

Henkeh  Th.,  518 

Henneberg,  W.,  397 

Henninger,  A.,  60 

Henocque,  A.,  219 

Henri,  V.,  enzymes,  13,  14;     oxyhsemo- 
globin,  199 

Henriques,  R.,  138 

Henriques,  V.,  lecithin,  143;  lecithin- 
sugar,  184;  blood,  reducing  substance, 
240;  protein  regeneration,  417;  adi- 
pose tissue,  441 ;  fat  of  yolk,  504;  milk- 
fat,  538;  blood  gases,  697;  lungs,  oxi- 
dation, 704 

Hensen,  V.,  lymph,  253;  lymph-gases, 
702 

Henze,  M.,  gorgonin,  82;  iodogorgonic 
acid,  82;  aspartic  acid,  88;  hsemocy- 
anin,  219;  copper  in  cephalopods,  286; 
spongosterin,  337;  extractives  of  mus- 
cle, 455;   muscles  of  octopods,  478 

Heptner.  F.  K.,  327 

THeritier,  534 

Herlant,  L.,  nucleic  acids,  154-156 

Hermann,     L.,    muscle    work,     9,     466; 
haemoglobin  in  starvation,  244;    forma- 
tion ot  fa?ces,  409;    muscle  rigor,  466; 
allantom,  583 
eron,  J.,  amylolysis,  128,  344;    invertin 

Bin  the  intestine,  378 


Herrmann,  Aug.,  trypsin,  394;  uric  acid, 
569 

Herrmann,  E..  166 

Herter,  E.,  saliva,  347;  ethereal  sulphuric 
acids,  589,  591,  637;  oxybenzoic  acids, 
636;   oxygen  tension,  706 

Herth,  R.,  proteosis  and  peptones,  52,  60 

Hervieux,  Ch.,  indol  and  indican,  186, 
592-594;  skatol  red  and  urorosein,  596; 
uroerythrin,  607 

Herzen,  A.,  spleen  and  digestion,  274; 
secretion  of  gastric  juice,  350;  charging 
theory,  365-385;  pancreatic  juice,  384, 
385 

Herzfeld,  A.,  107 

Herzog,  R.  O.,  zymases,  11;   histidine,  100 

Hesse,  A.,  296 

Hetper,  J.,  htemin,  212;    ha'mopyrol,  215 

Heubner,  O.,  casein,  utilization,  530; 
nurslings  metabolism,  752 

Heubner,  W.,  fibrinogen,  177;  mytolin, 
451 

Heiiss,  E.,  693 

Hewlett,  A.  W.,  427 

Hewlett,  R.  T.,  40 

Hewson,  W.,  226 

Heymann,  F.,  pseudomucin,  500 

Heymans,  J.  F.,  481 

Heynsius,  A.,  salting  out  of  proteins,  51; 
bilicyanin,  322,  324 

Hiestand,  O.,  144 

Hilbert,  P.,  634 

Hildebrandt,  H.,  oxalic  acid,  583;  amino- 
benzoic  acid,  636;  conjugations  with 
glucuronic  acid,  638 

Hildebrandt,  P.,  mammary  glands,  514, 
537;  conjugations  with  glucuronic  acid, 
610 

Hildesheim,  764 

Hilger,  69 

Hilger,  A.,  458 

Hiller,  E.,  438 

Hirsch,  R.,  glycolysis,  303;  amino-acids  in 
the  urine,  614 

Hirschberg,  A.,  222 

Hirschfeld,  E.,  371 

Hirschfeld,  F.,  work  and  nitrogen  excre- 
tion, 471;  uric  acid,  569;  acetone  bodies, 
669;  metabolism,  protein,  738,  750,  765; 
nutritive  requirements,  770 

Hirschfeld,  H.,  643 

Hirschl,  J.  A.,  658 

Hirschler,  A.,  putrefaction  processes  in  the 
intestine,  405,  407,  589;  ethereal 
sulphuric  acids,  589 

His,  W.,  434 

His,  W.,  Jr.,  pvu-ine  bases,  164;  uric  acid, 
575;  pyridine,  639 

Hlasiwetz,  H.,  protein  bodies,  29;  amino- 
acids,  87,  88,  91 

Hochhans,  H.,  245 

Hoeber,  R.,  colloids,  169;  osmotic  pres- 
sure, 189;  determination  of  alkalinity, 
191;     permeability    of    the    blood-cor- 


INDEX  TO  AUTHORS. 


825 


puscles,  195;  of  the  muscles,  465; 
absorption,  427;  acidity  of  the  urine, 
545 

Hone,  J.,  652 

Hofbauer,  L.,  343 

van't  Hoff,  J.,  6 

Hoffmann,  A.,  586 

HofTmann,  F.  A.,  transudates,  259,  261; 
oedematoas  fluid,  265;  blood-sugar, 
297;    diabetes,  299 

Hoffmann,  J.,  544 

Hoffmann,  P.,  626 

Hofmann,  tyrosine  test,  91 

Hofmann,  Fr.,  fat  and  fat  formation,  441- 
443 

Hofmann,  K.  B.,  muscles,  474;   brain,  486 

Hofmeister,  F.,  cellular  enzymes,  23;  pro- 
tein nitrogen,  27;  halogen  proteins,  30; 
glucosamine  in  proteins,  33;  amino- 
acids,  3;  bindings,  34;  removal  of  pro- 
teins, 44;  synproteose,  55;  collagen, 
77;  gelatine,  79;  serglobulin,  178,  180; 
pus,  267;  gastric  movements,  366; 
protein  absorption,  414,  416;  assimi- 
lation limit,  420;  ovalbumin,  507; 
crystallization  of  proteins,  507;  urea 
formation,  553;  creatinine,  564;  glo- 
bulin in  urine,  646;    lactose,  666 

Hofmeister,  V.,  gastric  digestion,  370; 
cellulose,  397;    protein  absorption,  413 

Holde,  D.,  132 

Holmgren,  E.  S.,  submaxillary  glands, 
340;   phenylhydrazine  test,  658 

Holmgren,  Fr.,  blood-gases,  697;  elimi- 
nation of  carbon  dioxide,  710 

Holmgren,  J.,  muscle  stroma,  448,  451 

V.  Hoist,  G.,  mucins,  66,  265;  transudates, 
258 

V.  Hoogenhuyze,  C.  J.,  creatinine,  563, 
567 

Hooker,  D.,  255 

Hopkins,  F.  G.,  halogen  proteins,  30; 
Adamkiewicz's  reaction,  42;  trypto- 
phane, 102,  103;  protein  crystallization, 
182,  507,  508;  uric  acid,  569,  577;  uro- 
bilin, 603,  605,  606;  butterflies.  690 

Hoppe-Seyler,  F.,  oxidations,  3;  cleavage 
processes,  5;  proteins,  36,  47;  compound 
proteids,  65;  collagen,  76;  protoplasm, 
141,  145,  148;  lecithins,  143,  145,  147; 
glycogen,  148,  287;  cholesterin,  148; 
nuclein,  149;  xanthine,  160;  blood- 
plasma,  187;  blood-corpuscles,  194, 
219;  blood-pigments,  190,  197,  198,202, 
204-210,213,214,  217;  urobilinoids,  214, 
603;  blood  analysis.  237;  chyle,  251; 
pericardic  fluid,  260;  pus,  267-269; 
cerebrin,  268;  struma,  275;  bile,  326, 
329;  excretin,  410;  cartilage,  434:  bones 
and  teeth,  437,  440;  lactic  acids.  461, 
463,  469,  608;  retina,  489;  ovovitellins, 
47,  503;  casein,  518;  milk,  526;  bile 
acids  in  urine,  652;  inosite,  668; 
chitosan,  687;  sebum,  691;  respiration 
apparatus,  712,  722 


Hoppe-Seyler,  G.,  determination  of  blood 
pigments,  217;  elimination  of  phenol, 
590;  of  indoxyl,  592-595;  urobilm, 
604,  606 

Horbaczewski,  J.,  keratin,  73;  elastin, 
75,  76;  glutamic  acid,  88;  purine  bases, 
159,  160;  uric  acid,  568;  formation  of 
uric  acid,  274,  570;  urostealith,  682; 
metabolism,  739 

Hombcrg,  A.  J.,  gastric  juice,  352,  353 

Home,  R.  M.,  171 

Horodynski,  W.,  241 

Hoagardy,  A.,  181 

Howell,  W.  H.,  464 

Huber,  A.,  blood  coagulation,  171;  fibrin- 
olysis, 174;    liver  diastase,  295 

Huebner,  R.,  403 

Hiifner,  G.,  leucine,  85;  blood-pigments, 
197,  198,  199,  203-200,  209,  218; 
spectrophotometry,  218;  dissociation 
of  oxy haemoglobin,  199,  704;  glyco- 
cholic  acid,  313;  bile,  313;  bird's  eggs, 
gases  of,  509;  urea  determination,  562; 
oxygen  tension,  706;  gases  of  the 
swimming-bladder,  711 

Hiirtlile,  K.,  183 

Hugounenq,  L.,  biliverdin,  324;  haema- 
togen,  503;   ash  of  child  and  milk,  535 

Huiskamp,  W.,  fibrinogen  and  fibrin  for- 
mation, 173,  174,  175,  176,  177;  nucleo- 
proteids,  178,270,  271;  thymus  histones, 
270,    271 

Hultgren,  E.  O.,  foodstuffs,  417,  421; 
appendix,  427;    diets,   764,   767 

Humnicki,  V.,  koprosterin,  337 

Hundeshagen,  F.,  sponges,  81;  lecithin,  143 

Hupfer,  Fr.,  586 

Huppert,  H.,  digestion  products,  55; 
glycogen,  221,  288;  reaction  for  bile 
pigments,  322,  653;  estimation  of 
pepsin,  357;  meat,  476;  urea,  555;  uro- 
leucic  acid,  600;  urine,  estimation  of 
proteins,  646;  laiose,  665;  determin- 
ation of  acetone,  673 

Hurtley,  W.  H.,  600 

Husson,  539 

Hybbinette,  S.,  608 

Ide,  457 

Ignatowski,  A.,  675 

Ikeda,  K,  14 

Inagaki,  C.,  181 

Inoko,  Y.,  198 

Inonye,   K.,   intestine  nucleic  acid,    151, 

154;  cytosine,  165;  lacticacidinurine,608 
Inonye,  Z.,  364 

Irisawa,  T.,  lactic  acid,  241,  608 
Ito,  M.,  643 
Iversen,   A.,   secretion  of  prostate,   496; 

concretions,  498 
Iwanoff,  621 
Iwanoff,  L.,  nucleases,  153,  394 

Jaarsveld,  G.  J.,  588 
Jablonsky,  J.   387 


826 


INDEX  TO  AUTHORS. 


Jackson,  H.  C,  lactic-acid  formation,  461; 
kynurenic  acid,  600 

Jacobsen,  A.,  184 

Jacobsen,  O.,  human  bile,  326,  327;  be- 
havior of  aromatic  substances  in  the 
body,  635 

Jacobson,  J.,  19 

Jacoby,  M.,  oxidation  enzymes,  8,  18; 
autolysis,  22,  281,  713;  phosphorus 
poisoning,  blood  in,  172,  175;  demoli- 
tion of  uric  acid,  574 

Jacubowitsch,  347 

Jaeckle,  H.,  human  fat,  132,  134,  441;  fat 
determinations,  136;  lipomia,  442 

Jiiderholm,  A.,  blood  pigments,  204, 
209 

Jaeger,  A.,  711 

Jaffa,  M.  E.,.  diets,  767,  769 

Jaffe,  M.,  ornithin  and  ornithuric  acid,  97, 
635;  bile-pigments,  322;  creatine,  456; 
phenylsemicarbazide,  556;  urethane, 
563;  creatinine,  565,  567;  uric  acid, 
572,  575;  urinary  indican,  592-594; 
kynurenic  acid,  600;  urobilin,  409,  601, 
602,  604,  605;  behavior  of  aromatic 
substances,  633,  635-638;  furfurol,  637; 
thriophenic  acid,  637 

V.  Jaksch,  R.,  alkalinity  of  blood,  223; 
urea  in  the  blood,  240;  brain,  480; 
urine,  partition  of  nitrogen  in,  549; 
volatile  fatty  acids,  608;  melanin,  651; 
phenylhydrazine  test,  657;  pentosuria, 
666;    acetone,  668 

J.akowsky,  M.,  400 

Jalowetz,  E.,  125 

Jamieson,  G.  S.,  82 

Jantzen,  F.,  537 

Jaquet,  A.,  oxidation  enzymes,  8;  hsemo- 
globin,  198;  estimation  of  blood  pig- 
ments, 219 

Jastrowitz,  M.,  pentoses,  110,  666 

Jelinek,  J.,  fermentation,  21,  461 

Jensen,  P.,  459 

Jerome,  W.  Smith,  612 

Jesner,  S.,  491 

Joachim,  J,,  serglobulin,  179;  transuda- 
tes, 2^8,  261,  263 

Jodlbauer,  436 

Johansson,  J.  E.,  serglobulin,  182;  ex- 
change of  material  and  gas,  474,  759- 
762,  768 

Johnson,  E.  G.,  chymosin,  361 

Johnson,  St.,  creatinine,  563-566 

Johnson,  T.  B.,  triticonucleic  acid,  150; 
cytosine,  165 

John,  S.,  pig-bile,  313;  elimination  of 
ammonia,  624 

JoUes,  A.,  blood-catalases,  20;  oxidation 
of  protein,  32;  determination  of  iron, 
217;  bile-pigments,  321,  653;  milk,  533; 
urinometer,  547;  uric  acid,  578;  uro- 
bilins, 602;  urine,  iron  of,  626;  protein 
of,  642;   nucleohistone,  647 

Joly,  5.39 

Jolyet,  697 


Jones,  W.,  autolysis,  22;  nucleoproteids, 
72;  nucleic  acids,  153;  enzymes  of 
nucleic  acids,  158,  273,  571;  thymine, 
165;  nucleases,  271;  thymus,  271; 
spleen,  and  uric  acid,  273,  275 

de  Jonge,  D.,  693 

Jorissen,  W.  P.,  6 

Jornara,  D.,  693 

Josephsohn,  A.,  600 

Jowett,  H.  A.  D.,  278 

Junger,  E.,  318 

JungHeisch,  E.,  463 

Justus,  J.,  166 

Juvalta,  N.,  633 

Kalberlah,  F.,  670 

Kanitz,  A.,  steapsin,  389;   trypsin,  393 

Kareff,  N.,  173 

Kast,  A.,  intestinal  putrefaction,  407; 
virine  sulphur,  611;  elimination  of 
chlorine,  616;  methane  substituted  by 
halogens,  631;  demolition  of  sulphur- 
ized substances,  632;    sweat,  694 

Kastle,  J.  H.„enzymes,  7,  8,  16 

Katsuyama,  K.,  lactic  acid,  241,  461; 
inanition,  731 

Katz,  A.,  604 

Katz,  J.,  477 

Katzenstein,  A.,630 

Katzenstein,  G.,  760 

Kauder,  G.,  globulin,  ISO;  seralbumin, 
181 

Kaufmann,  M.,  glycogen,  295;  sugar  of 
the  blood,  297;  svigar  formation  from 
fat,  306;  fat  formation,  444-446; 
respiratory  quotient,  446;  muscles, 
urea  of,  455j  sugar  consumption  of, 
468;  virea  formation,  5'54;  methods  of 
metabolism  investigations,  723 

KaufmanUj  M.,  urine  purines,  579 

Kaufmann,  Martin,  hunger,  protein  cata- 
bolism  in,  730;  gelatine,  nutritive  value 
of,  746 

Kaup,  J.,  471 

Kausch,  W.,  293 

Keller,  A.,  614 

Keller,  W.,  635 

Kellner,  O.,  toodstuffs,  utilization  of,  418; 
work  and  protein,  471;  asparagin,  747; 
diets,  764 

Kelly,  A.,  extractives  of  muscles,  455; 
chitin,  686 

Kermauner,  F.,  409 

Kerner,  G.,  564 

Kiliani,  H.,  105 

Kirschmami,  J.,  746 

Kirk,  R.,  600 

Kisch,  F.,  459 

Kishi,  K.,  277 

Kistermann,  658 

Kitagana,  F.,  485 

Kjildahl,  nitrogen  determination,  556, 
557 

Hages,  A.,  318 

V.  Klaveren,  H.  K.  L..  208 


INDEX  TO  AUTHORS. 


827 


Ivlecki,  K,  409 

Kleine,  F.,  urinary  sulphur,  611;  formani- 
lide,  63'4 

Klemensiewicz,  R.,  stomach,  formation  of 
acid,  363;  pylorus  secretion,  365 

Klemperer,  G.,  chymosin,  361;  uro- 
chrome,  601 ;  oxalic  acid,  behavior  of, 
630;  metabolism  of  protein,  73S,  750,  765 

af  ICIercker,  O.,  563 

Klingemann,  F.,  539 

Klingenberg,  K.,  634 

Klug,  F.,  tryptophane,  102;  pepsin  and 
pseudopepsin,  354;  products  of  di- 
gestion, 360;  trypsin,  390;  elimination 
of  phosphoric  acid,  620 

V.  Knalli-Lenz,  E.,  286 

Knapp,  K.,  sugar  test,  116;  determination 
of  sugar,  660,  662 

V.  Knieriem,  W.,  digestion  of  cellulose, 
397;  formation  of  urea,  550,  551;  for- 
mation of  vu'ic  acid,  572 

Knopfelmacher,  W.,  human  fat,  132,  441 

Knoop,  F.,  histidine,  99;  methyl  imi- 
dazole, 108;  proteoses  in  the  blood,  183, 
415;  protein  regeneration,  416;  demoli- 
tion of  aromatic  substances,  633,  634 

Knop,  W.,  562 

Kobert,  H.  U.,  196 

Kobert,  R.,  haemolysis,  193;  cyanmet- 
hiemoglobin,  205;  urinary  iron,  626; 
melaiiines,  6S9 

Kobrak,  E.,  531 

Koch,  W.,  lecithans  and  lecithins,  146 
148,  487,  488;  neurokeratin,  480 
protagon,  482;  cephalin,  485,  486,  488 
cholesterin,  480;  brain  analysis,  487 
milk,  532 

Kocher,  Th.,  276 

Kochs,  W.,  ethereal  sulphuric  acids,  280; 
hippuric  acid,  586 

Koefoed,  E.,  518 

Kohler,  A.,  meat  476;  calorific  value, 
727;  asparagin,  447 

Konig,  J.,  muscle,  475;  milk,  527-529, 
638;  foods,  773 

Koppe,  H.,  blood  corpuscles,  192-196, 
220;  volume  determination,  237;  iso- 
tomy,  192,  193;  haemoglobin,  193,  194; 
secretion  of  hydrochloric  acid,  364 

Koster,  H.,  521 

Koettgen,  E.,  490 

Kohlrausch,  Fr.,  191 

Kolisch,  R.,  blood,  240;  creatinine,  567; 
urinary   histone,    647 

Koraen,  G.,  muscular  activity,  474;  food 
and  metabolism,  762 

v.  Koranyi,  A.,  .  blood,  224;  osmotic 
pressure,  256 

Kossel,  A.,  nitrogen  of  the  proteins,  27; 
protein  hydrolysis,  29,  78;  arginase, 
36,  97,  550;  protamines,  59,  61-64,  84, 
96,  98,  498;  histone.  62,  63,  96,  97; 
nucleoproteins,  71,  72;  aminovaler- 
ianic  acid,  84;  serine,  95;  hexone  bases, 
96,  98-100;    a-proline,  101;   saponifica- 


tion, 136;  the  cell,  140,  141,  142;  nucleo- 
histone,  141,  270:  nucleic  acitls,  152- 
156,  498;  pyrimidine  group,  152,  164- 
166;  pi  isminic  arid,  l.)6;  purine  bases, 
152,  156,  158,  161-164,  271,  284,  382, 
454;  haemoglobin,  197;  blood  plates, 
222;  carebrosides,  268,  482-485;  pro- 
tagon, 431,  4S2;  ichtlmlin,  504 

Kossler,  A.,  phenol  determination,  589, 
590 

Kostin,  S.,  206 

Kostytschew,  S.,  nucleic  acid,  153,  155 

Kotake,  Y.,  nucleic  acid  of  the  intes- 
tine, 151,  154;  cytosine,  165;  vanillin, 
635 

Kotliar,  E.,  280 

Kowalewsky,  K.,  products  of  digestion, 
53;    uric  acid,  572 

Kowarski,  A.,  455 

Kramm,  W.,  565 

Kranenburg,  W.  R.  H.,  364 

Krarup,  J.  C,  761 

Kratter,  Jul.,  442 

Kraus,  Fr.,  fatty  liver,  283;  amino  acids 
and  formation  of  carbohydrates,  305 

Krauss,  E.,  594,  595 

Krawkow,  N.  P.,  amyloid,  69,  70,  431; 
chitin,  686,  687 

Krehl,  L.,  647 

Kreis,  H.,  132 

Kresteff,  S.,  365 

Kreusler,  W.,  protein  substances,  29 

Kreuzhage,  C,  471 

Krieger,  H.,  protein  substances,  38;  seral- 
bumin, 181,  182;    phcsphaturia,  621 

Krimberg,  R.,  457 

Krober,  E.,  Ill 

Kronig,  B.,  168 

Krogh,  A.,  oxygen  in  the  blood,  704,  711 

Kronecker,  F.,  588 

Kriiger,  A.,  denaturization  of  proteids, 
40;  haemoglobin,  201;  leucocytes,  221; 
blood,  221,241,242;  spleen,  274;  liver, 
286;  saliva,  sulphocyanide,  343;  intes- 
tinal juice,  377 

Kriiger,  M.,  saponification,  136;  alloxuric 
bases,  156,  159,  163;  in  fieces,  408;  in 
urine,  578-581;   ammonia,  625 

Kriiger,  Th.  R.,  peptones,  58,  80;  phospho- 
carnic  acid,  457,  474 

Krug,  B.,  752 

Krukenberg,  F.  C.  W.,  hyalogens,  68; 
keratin  proteoses,  73;  skeletins,  81,  82 
lipochromes,  186;  haemerythrin,  219; 
extractive  bodies  of  muscle,  455,  456; 
bird's  eggs,  509;  urostealith,  682;  bird's 
feathers".' 690 

Krummacher,  O.,  determination  of  fat, 
137;  activity,  work,  metabolism,  471; 
heat  of  combustion,  728;  gelatine,  its 
nutritive  viTue,  746 

Krumbholz,  C.  J.,  422 

Kiibel.  F. ,  saliva,  345,  346 

Kiihling,  O.,  behavior  of  aromatic  sub- 
stances, 637,  638 


828 


INDEX  TO  AUTHORS. 


Kiihne,  W.,  enzymes,  10;  proteose  and 
peptones,  51-60,  359;  gelatine,  78;  ty- 
rosine, 89;  paraglobiilin,  178;  hsema- 
toidin  and  lutein,  216;  glycogen,  287; 
gastric  digestion,  367,  371 ;  the  pancreas 
and  its  enzymes,  387-391,  395,  396; 
emulsion  of  fat,  422;  proteins  of  the 
muscles,  448,  449;  smooth  muscles, 
477,  478;  neurokeratin,  73,  480,  489; 
pigments  of  the  eye,  490,  491;  lens,  493; 
corpora  lutea,  498 

Kiilz,  C,  259 

Kiilz,  E.,  cystine,  92;  pentoses,  110,  666; 
amylolysis,  125,  344,  389;  glycogen, 
287,  288,  291,  295;  diabetes,  298,  300; 
gastric  juice,  364;  muscle  activity,  468; 
gases  in  milk,  533;  conjugated  glu- 
curonic acids,  632,  638;  oxybutyric 
acid,  673 

Kiilz,  R.,  methffimoglobin,  204;  deter- 
mination of  glycogen,  290;  gases  of  the 
saliva,  342,  702 

Kueny,  L.,  117 

Kiister,  W.,  blood-pigments,  209-212; 
hsematinic  acids,  211,  320;  haemo- 
pyrrol,  214;  bile-pigments,  210,  320, 
321,  323,  324 

Kuhn,  491 

Kuliabko,  A.,  pancreas,  302;    ureine,  563 

Kumagawa,  ]\I.,  formation  of  fat,  444; 
determination  of  sugar,  663;  protein 
metabolism,  738,  750,  765 

Kunkel,  A.  J.,  arsenic,  166;  carbon- 
monoxide  blood-test,  206;  iron  prepar- 
ations, 245;  iron  and  bile,  326,  332; 
urinary  iron,  626 

Kuprianow,  J.,  460 

KurajefT,  D.,  halogenic  proteins,  30;  plas- 
teine,  56;  protamines,  63;  tryptophane, 
102 

Kurbatoff,  D.,  135 

Kurpjuweit,  O.,  407 

Kusmine,  K.,  255 

Kutscher,  Fr.,  nitrogen  of  the  proteins, 
27;  products  of  digestion,  54,  57;  his- 
tones,  61,  63,  88,  89;  protein  hydrolysis, 
78,  88,  89;  hexone  bases,  96-100;  nu- 
cleic acids,  154;  martamic  acid,  154; 
cytosine,  166;  thymine,  271;  erepsin, 
380;  guanidine,  394;  choline,  394;  intes- 
tinal contents,  400;  absorption  of  pro- 
tein, 413;  bases  of  meat  extracts,  457; 
gelatine,  oxidation,  582;  neurine,  615 

Kuwschinski,  P.,  387 

Kyes,  P.,  lecithin,  146;  in  hsemolysis,  193 


Laache,  S.,  246 

V.  Laar,  J.,  73 

Ladenburg,  A.,  496 

Laidlaw,    P.    P.,    blood-pigments,    216; 

turacin,  690 
Lanceraux,  301 
Landauer,  A.,  bile  and  putrefaction,  406; 

water  and  metabolism,  734,  754 


Landergren,  E.,  foodstuffs,  417,  421; 
metabolism,  730,  738,  748;  sugar  forma- 
tion, 749;    diets,  764,  767 

Landois,  L.,  stroma  fibrin,  195;  trans- 
fusion, 248 

Landolt,  H.,  688 

I^andwehr,  H.,  animal  gum,  67,  525,  608 

Lang,  G.,  646 

Lang,  J.,  taurine,  95;  lymph,  253 

Lang,  S.,  deamidation,  305;  lactic  acid 
formation,   572;     nitriles,   631 

de  Lange,  C,  milk,  533,  535 

Langendorff,  O.,  464 

Langer,  L.,  134 

Langgaard,  A.,  530 

Langhaus,  Th.,  216 

Langley,  J.  N.,  saliva,  345,  347;  propep- 
sin, 364;   cells  of  the  gastric  glands,  365 

Langstein,  L.,  carbohydrate  from  protein 
substances,  33,  65,  180,  181,  506;  diges- 
tion products,  53,  57;  scatosine,  103; 
proteoses  in  the  blood,  183,  415;  resid- 
ual nitrogen,  183;  serum,  188;  animo 
acids,  deamidation,  305;  ovoglobulin, 
506;  ovalbumin,  507;  ovomucoid,  508; 
alcaptonuria,  598,  600;  kynurenic  acid, 
600;  urinary  quotient,  628,  720;  lactic 
acid  in  the  urine,   665 

Lankester,  E,  R.,  219 

Lannois,  E.,  absorption  of  sugar,  419; 
cerumen,  592 

Lapicque,  L.,  spleen,  274;  liver,  285,  286; 
diets,  767 

Lappe,  J.,  379 

Laptschinsky,  M.,  493 

Laqueur,  E.,  casein  and  rennet  coagula- 
tion, 518-521;  paracasein,  521;  lipase, 
363 

Larin,  A.  M.,  358 

Lassaigne,  J.  L.,  583 

Lassar,  O  ,  223 

Lassar-Cohn,  bile  acids,  315,  317;  human 
bile,  318;  myristic  acid  in  the  bile,  325 

Latarjet,  A.,  352 

Latham,  4 

Latschenberger,  J.,  blood,  170;  bile-pig- 
ments, 330,  332;  iron  of  liver,  332; 
protein  absorption,  413 

Latschinoff,  P.,  bile-acids,  317 

Laulanie,  F.,  respiratory  quotient,  446; 
work  and  sugar  consumption,  473,  760 

de  Laval,  G.,  526 

Laves,  E.,  diabetes,  300;    milk-fat,  530 

Laves,  M.,  295 

Lavoisier,  A.  L.,  759 

Lawrow,  D.,  digestion  products,  53; 
histones,  61;  histidine,  99;  blood-pig- 
ments, 208;  conjugated  glucuronic 
acids,  638 

Lawrow,  Maria,  56 

Lawes,  445 

Laxa,  O.,  casein  and  rennet  coagulation, 
519,521 

Lazarus-Barlow,  W.  S.,  255 

Lea,  Sh.,  346 


INDEX  TO  AUTHORS. 


829 


Leathes,  J.  B.,  autolysis  of  spleen,  273: 
fat  formation,  297;  proteosis,  absorp- 
tion of,  415,  417;  ovarial  fluid,  500 

Lebedeff,  A.,  liver  fat,  282;  fat  of  food,  442 

Leclerc,  A.,  694 

Leconte,  P.,  351 

Ledderhose,  G.,  glucosamine,  120;  chitin, 
686 

Ledoux,  A.,  234 

van  Leersum,  E.  C,  pentoses,  112;  con- 
jugated glucuronic  acids,  122 

Legal,  E.,  indol,  402;   acetone,  671 

Lehmann,  C,  fat  formation,  445;  meta- 
bolism in  starvation,  729;   in  work,  760 

Lehmann,  C.  G.,  saliva,  347;  protein  of 
egg-white,  506 

Lehinann,  Fr.,  695 

Lelimann,  K.  B.,  hgemorrhodin,  207; 
adipocere,  442;  sugar  determination, 
663 

Leichtenstern,  O.,  blood,  243,  244 

Leick,  441 

Leipziger,  R.,  719 

Lelli,  G.,  480 

Lemaire,  F.,  isomaltose,  608;  and  lactose 
in  the  urine,  665 

Lemus,  W.,  518 

Leo,  H.,  fatty  liver,  282;  diabetes,  300; 
acids  of  gastric  juice,  375,  376;  fatty 
degeneration,  443;  laiose,  665;  nitrogen 
deficit,  718 

Lepage,  L.,  384 

Lepine,  R.,  conjugated  glucuronic  acids, 
122;  pentoses,  184;  virtual  sugar,  184; 
glycolysis,  185,  251,  302;  glycogen,  291; 
absorption,  419;  sulphur  of  urine,  611, 
612;  phosphorus  compounds  in  the 
urine,  614;  urine  poison,  615;  maltose 
in  the  urine,  667 

Lepinois,  E.,  616 

Lerch,  493 

Lesem,  W.,  482 

Lesnik,  M.,  633 

Lesser.  1^.  J.,  417 

Lesser,  K.  A.,  254 

Leube,  W.,  urine,  640;   sweat,  694 

Leuchs,  H.,  serin,  95;  oxypyrrolidine  car- 
boxvlic  acid,  102;  glucosamine,  107, 
108,'  120 

Levene,  P.  A.,  autolysis,  22,  495;  pseudo- 
nucleic  acid,  46;  proteoses,  56;  tendon- 
mucin,  66,  428;  hydrolysis  of  gelatine, 
78,  79;  amino  acids,  83;  nucleic  acids, 
151-156,  514;  pjTimidine  bases,  152, 
154,  156,  394;  nucleoproteids,  272,  479; 
glucothionic  acids,  222,  273,  285,  431, 
514;  phlorhizin  diabetes,  298;  trj-psin, 
390;   ichthulin,  504;  dipeptides,  35 

Levites,  S.,  27 

Le^T,  A.  G.,  223 

Lev)%  H.,  637 

Le\j,  L.,  454 

Le^T.  ^I-  ^^9 

Lewandowski,  M.,  412 


Lewin,  K  ,  uric  acid  and  hippuric  acid, 
586;  indican,  593 

Lewin,  L.,  ha?moverdin,  208;  hydro- 
quinone  sulphuric  acid,  591 

Lewinskv,  J.,  187 

Lewis,  fh.,  478 

Lewy,  B.,  496 

Lewy.  Benno,  227 

V.  Leyden,  E.,  496 

V.  d.  Leyen,  E.,  593 

Lieben,  A.,  hsematoidin  and  lutein,  216; 
iodoform  test,  670 

Liebermann,  C,  cholesterin,  336;  shell  of 
bird's  eggs,  509;   carminic  acid,  690 

Liebermann,  L.,  catalases,  20;  protein 
and  acid  compounds,  38;  protein  re- 
action, 43;  pseudonucleins,  47,  151; 
lecithalbumins,  47,  349,  542;  fat 
determination,  137;  nucleins,  150,  151; 
mucosa  of  stomach,  349;  volk  of  egg, 
502,  504-506,  510,  511;  kidneys,  542; 
guaiac  test,  648 

V.  Liebig,  J.,  inosinic  acid,  155;  mineral 
bodies,  166;  fat  formation,  445;  work 
and  metabolism,  471,  472;  m'ea,  557; 
diets,  764 

Lieblein,  V.,  wound-secretion,  265;  liver 
and  urea  formation,  553 

Liebrecht,  A.,  30 

Liebreich,  O.,  neurine,  145;  protagon, 
481;   cholesterin  fat,  691 

Liepmann,  W.,  512 

Lifschutz,  J.,  isocholesterin,  337;  wool-fat, 
692 

Likhatscheff,  A.,  637 

Lilienfeld,  L.,  histone,  62,  270;  nucleo- 
histone,  141,  270;  cell  nucleus,  142; 
fibrin  ferment  and  blood  coagulation, 
176,227;  blood-plates,  222, 227:  thymus, 
271.272;   leuconuclein,  229,  270 

V.  Limbeck,  R.,  223 

Limpricht,  H.,  455 

Lindberger,  W.,  tryptic  digestion,  393; 
bile  and  putrefaction,  406 

Lindemann,  L.,  Ill 

V.  Linden,  M.,  690 

Lindvall,  V.,  73 

Ling,  A.  R.,  125 

Lingle,  D.  J.,  464 

Linossier,  G.,  543 

Linser,  P.,  sebum,  691 

Lintner.  C.  J..  128,  129 

Lintwarew,  S.  J.,  pyloric  reflex,'  368; 
pancreatic  juice,  383 

Lipliawskv,  A.,  672 

Lipp,  A.,  89 

Lippich,  Fr.,  563 

V.  Lippmann,  E.  O.,  enzjTnes,  21; 
tyrosine,  90;   carbohydrates,  105 

Lister,  J.,  226 

Lloyd- Jones.  E.,  222 

Loeb,  J.,  ion  action,  166,  168,  169;  mus- 
cles, 464:    metabolism,  761 

Loeb,  L.,  blood  coagulation,  232,  235 


830 


INDEX  TO   AUTHORS. 


Loeb,  W.,  carbon-dioxide  assimilation,  1; 

muscle  work,  474 
Lobisch,   W.   F.,  bilipurpurin,  324;    con- 
nective tissue,  428 
Lobisch,  W.,  mammary  glands,  514;  case- 
in formation,  537 
LoUein,  W.,  pepsin  estimation,  357;  tryp- 
sin estimation,  392 
Lonnberg,  J.,  cartilage,  433;  kidneys,  542 
Lonnquist,  B.,  351 
Lore  her,  G.,  361 
Loeschcke,  K.,  289 
Loevenhart,  A.  R.,  enzymes,  7, 17,  446 
Loew,  O.,  active  proteins,  4;    catalases, 
7,   20;   protein  nitrogen,   27;    proteins, 
30,  34,  49;    sugar  syntheses,  114;    cell 
nucleus,  167 
Loewenthal,  W.,  398 

Loewi,  O.,  phlorhizin  diabetes,  298;  sugar 
formation,  306;  protein  legeneration, 
416;  urea  formation,  550;  uric-acid 
formation,  571;  allantoin,  584;  con- 
jvigated  glucvironic  acids,  610;  phos- 
phorus metabolism,  619,  719 
L6\Yit,  M.,  227 

Loewy,  A.,  diamines,  97,  98;  alkalinity 
of  the  blood,  223;  high  altitudes,  246, 
614,  762;  workandmetabolism  with, 471, 
760;  amino  acids  in  the  urine,  614, 
oxygen  absorption  and  pressure,  704, 
705;  metabolism,  762 
Lohmann,  lysine,  99;  choline,  394;  neurine, 

615 
Lohnstein,  Th.,  urometer,  547;    saccharo- 
meter,  657,  664;    sugar  estimation,  664 
Lohrisch,  H.,  397 
Lombroso,  421 
London.  E.  S.,  digestion,  370,  400,  401; 

blood  in  starvation,  732 
Long,  J.  H.,  casein,  518;  urine  coefficient, 

627 
Lorrain  Smith,  J.,  chlorose,  246;  quantity 

of  blood,  248 
Lossen,  F.,  32 
Lubarsky,  E.,  138 
Luchsinger,  B.,  glycogen,  291,  293;  sweat, 

693 
Luciani,  L.,  starvation,  244,  728 
Ludwig,     C.,     pseudohaemoglobin,     203; 
formation   of   bile,   330;    gastric   diges- 
tion,  369;    pancreatic  juice,   382;    ab- 
sorption of  protein,  414;    of  sugar,  420; 
blood-gases,  696,  697;  temperature  and 
metabolism,  761 
Ludwig,  E.,  fat  of  demoid  cysts,  138,  502; 

uric  acid  estimation,  576 
Lucke,    A.,    hyalin,    69,    687;     pus,    269; 

benzoic  acid  reaction,  587 
Liidecke,  K.,  glycerophosphoric  acid,  144 
Liithje,    H.,    sugar    formation,    303-305; 

oxalic  acid,  582 
Liittke,  J.,  376 
Lukjanow,  S.,  bile,  307,  308;    starvation, 

732 
Lummert,  W.,  fat,  283;  fat  formation,  445 


Lunin,  N.,  requirement  for  mineral  bodies, 
736;    artificial  feeding,  739 

Lusk,  Graham,  phlorhizen  diabetes,  298, 
462:  sugar  formation,  306;  lactose  in 
the  intestine,  419;  lactic-acid  forma- 
tion, 462;    fermentation  of  sugar,  665 

Lussana,  F.,  295 

Luther,  E.,  659 

Luzzatto,  A.,  583 

Maas,  O.,  49 

MacuUum,  A.  B.,    potassium,    occurre/ce 

of,  167,  464;   iron  preparations,  245 
Maccadam,  J.,  471 

Mac  Galium,  J.  B.,  378 

Macfayden,  A.,  fermentation,  10;  intes- 
tinal contents,  399 

V.  Mach,  W.,  572 

Mackay,  J.  C.  H.,  652 

Macleod,  J.,  phosphocarnic  acid,  457,  470; 
carbamic  acid,  563 

Mac  Munn,  Ch.  A.,  hfematoporphyrin, 
213,  649,  690;  urobilinoids,  214,  603; 
echinochron,  219;  cholohje matin,  324; 
myoha?matin,  454;  tetronerythyrin,  690 

Madsen,  Th.,  337 

Maetzke,  G.,  400 

Magnanini,  G.,  403 

Magnier,  626 

Magnus,  G.,  696 

Magnus,  R.,  284 

Magnus-Levy,  A.,  thyreoidea,  277;  liver, 
281;  diabetes,  306;  fat  formation,  445; 
hippuric  acid,  586;  volatile  fatty  acids 
in  the  urine,  608;  Bence-Jones's  pro- 
teid,  645;  acetone  bodies,  669,  674; 
respiration  experiments,  713,  722,  723; 
metabolism,  757,  758,  762 

Maillard,  L.  C,  594 

Maillard,  M.  L.,  168 

Majert,  W.,  496 

Makris,  C.,  530 

Malcolm,  J.,  619 

Malengreau,  F.,  thymus  and  nucleohis- 
tone,  270,  271 

Malfatti,  H.,  tryptophan,  54;  purine 
bases,  581 

Mall,  F.,  80 

Mallevre,  A.,  397 

Maly,  R.,  oxyprotic  acids,  31:  peptones, 
60,  746;  bile-pigments,  320-323;  hy- 
drobilirubin,  320,  603;  saliva,  341; 
secretion  of  hydrochloric  acid,  364; 
pancreatic  juice,  387;  bile  and  putrefac- 
tion, 406;  luteines,  505;  creatinine,  566 

Manasse,  A.  92 

Manasse,  P.,  283 

Manche,  M.,  648 

Manchot,  W.,  oxidations,  6,  7,  19 

Mandel,  A.  R.,  lactic  acid  formation,  462 

]\Iandel,  J.  A.,  nucleic  acids,  152,  153,  .154, 
514;  spleen  nucleoproteid,  272;  glu- 
cothionic  acids,  222,  272,  273,  285,  514; 
mammary  gland,  514;  organic  phos- 
phorus in  the  urine,  614 


INDEX  TO  AUTHORS 


831 


Mandelstaram,  E.,  308 

Manicardi,  C,  457 

Manning,  T.  D.,  378 

Mansfield,  G.,  264 

Maqueiine,  L.,  starch,  126;   inosite,  458 

Murcet,  xanthine,  159;  excretin,  410; 
excretolic  acid,  410 

Marchetti,  G.,  442 

Marclilewski,  L.,  pigments  of  leaves  and 
blood,  197,  214;  h»min,  212;  hsemo- 
pyrrol,  214;  phylloerythrin  and  cholo- 
liBematin,  324 

Marcus,  E.,  serglobulins,  178 

Marcuse,  G.,  phosphorus  metabolism,  719 

Marcuse,  W.,  glycogen,  468;  lactic  cid 
formation,  469 

Mares,  Fr.,  569 

Marfori,  P.,  630 

Margulies,  658 

Mark,  H.,  283 

Marquardsen,  E.,  407 

Marshall,  J.,  597 

Martin,  C.  J.,  fibrin  formation,  176;  blood 
coagulation,  234 

Martin,  S.  H.,  397 

Martins,  F.,  376 

Martz,  692 

Maschke,  O.,  protein  crystals,  38;  crea- 
tinine, 565 

Masius,  J.  B.,  stercobilin,  320,  409 

Masloff,  A.,  377 

Massen,  V.,  552 

Masuvama,  M.,  503 

Mathieu,  E.,  697 

Mathews,  A.,  arbacine,  62;  protamines, 
63,  498;  lysine,  98;  nucleic  acids,  154, 
498;   ion  action,  169;    fibrinogen,  172 

Matthes,  M.,  intestinal  contents,  407; 
urinary  pepsin,  615;  urinary  histone, 
647 

Maurenbrecher,  A.  D.,  113 

Mauthner,  J.,  cholesterin.  334;  asparagin, 
1 ;    nutritive  value  of,  747 

Maximowitsch,  S.,  seralbumin,  181,  182 

May,  R.,  Ill 

Mayer,  A.,  626 

Maver,  J.,  753 

Mayer,  L.,  418 

Mayer,  M.,  188 

Mayer,  P.,  cystine,  92-94;  mannoses,  108, 
109;  conjugated  glucuronic  acids,  122, 
123,  184,  609,  610,  667:  oxalic  acid,  583; 
indican,  593;  skatoxyl  glucuronic  acid, 
595 

Mayo-Robson,  A.  W.,  307 

Mays,  K.,  trj^psin;  390;   fuscin,  491 

Maze,  P.,  21 

Meara,  F.  S.,  52 

Medwedew,  A.,  oxidations,  818;  gly- 
cocholic  acid,  312 

V.  Mehring,  J.,  uiochloralic  acid,  122,  632; 
blood-sugar,  184;  portal  blood.  242, 
419;  glvcogen  formation,  291;  phlor- 
hizin  diabetes,  297,  298;  pancreas 
diabetes,    300;     amylolysis,    344,    388; 


assimilation  of  proteins,  412;  absorp- 
tion of  sugar,  420;  sarcosin,  631; 
acetonuria,  670 

Mehu,  C,  pleural  fluid,  261;  urobilin,  604, 
605 

Meillcre,  G.,  616 

Meinert,  C.  A.,  utilization  of  foodstuffs, 
418;  diets,  764 

Meinert z,  J.,  283 

Meisenheimer,  J.,  fermentation,  11,21 

Meissl,  F].,  445 

Meissl,  Th.,  513 

Meissner,  G.,  products  of  digestion,  359; 
egg-white,  506;  urea  formation,  553; 
allantoin,  583;   hippuric  acid,  585 

Meister,  V.,  553 

Mendel,  L.  B.,  lymph  formation,  255; 
alcohol,  369;  trypsinogcn,  385;  ab- 
sorption of  proteid,  412,  413,  414; 
muscle  extractives,  455;  uric  acid,  574; 
allantoin,  584;    kynurenic  acid,  600 

Mendes  de  Leon,  M.  A.,  531 

Menzies,  J.  A.,  205 

De  Merejkowski,  C,  690 

Mesernitzki,  502 

Messinger,  J.,  673 

Mester,  Br.,  intestinal  putrefaction,  407; 
indol  and  skatol,  596;  urinary  sulphur, 
611 

Mett,  S.,  estimation  of  pepsin,  357;  esti- 
mation of  trypsin,  392 

Meyer,  C,  286 

Meyer,  E..  blood-pigment  and  bile,  329; 
alcaptonuria,  598,  600;  nitrobenzene, 
635 

Meyer,  G.,  417 

Meyer,  H.,  camphor  glucuronic  acid,  122; 
adrenaline  substances,  278;  uric-acid 
formation,  572 

de  Mever,  J. .  302 

Michaelis,  H.,  185 

Michaelis,  L..  186 

Michel,  A.,  seralbumin,  181-183 

Micko.  K. ,  excrements,  409;  absorption 
of  casein,  719 

V.  Middendorff,  M.,  242 

Miescher,  F.,  protamines,  63,  64,  497; 
nucleins,  149,  152;  pus,  267,  269; 
sperm,  497;  salmon,  metabolism  of, 
738 

Milchner,  R.,  67 

Millon,  M.  E.,  protein  reaction,  30,  43; 
lactoprotein,  523 

Mills.  W.,  582 

Milroy,  J.  A..  209 

Milroy.  T.  H.,  nucleins,  150;  phosphorus 
metabolism,  619 

Minkowski,  O.,  blood  alkalinity.  223; 
ascites,  262;  glycogen.  295;  blood- 
sugar  and  liver,  297;  phlorhizin  dia- 
betes, 297,  298;  pancreas  diabete.-=.  300, 
301;  formation  of  bile  pigments.  331- 
333;  fat  absorption.  421;  pancreas  and 
absorption,  418,  425;  lactic  acid,  460, 
572,  573;  uric  acid  572,  573;  allantoin, 


832 


INDEX  TO   AUTHORS. 


584;  histozyme,  588;  acetone  bodies, 
670,  673,  674;   blood  in  diabetes,  702 

Mitjukoff,  K.,  500 

JVIittelbach,  F.,  fibrinogen,  173;  homo- 
gentisic  acid,  600;  estimation  of  pro- 
tein, 646 

Miura,  K.,  blood-sugar,  184;  glycogen 
formation,  290;  intestinal  invertase, 
379 

Miyamota,  S.,  355 

Mochizuki,  J.,  394 

Modrzejewski,  E.,  70 

MoUenberg,  R.,  243 

Moller,  J.,  409 

Morner,  C.  Th.,  membranins,  69,  434; 
albu  inoid,  74;  gelatine,  77,  79,  80. 
433,  434;  ichthylepidin,  81;  tyrosin 
test,  91;  vitreous  humor,  429,  491; 
cartilagenous  tissue,  430-433,  435; 
cornea,  434,  492;  bones,  436;  crystal- 
line lens,  492,  493;  ovomucoid,  508; 
perkaglobulin,  510;  homogentisic  acid, 
600;  estimation  of  chlorine,  619;  gallic 
and  tannic  acids,  637 

Morner,  K.  A.  H.,  sulphur  of  the  proteins, 
28;  cysteine  and  cystine,  28,  74,  92; 
thiolactic  acid,  28,  74,  94;  hydrolysis 
of  protein,  29;  albuminates,  49,  45; 
tyrosin,  89;  proteids  of  the  serum,  170- 
182;  ha^matin  and  ha?min,  212,  213; 
blister  fluid,  265;  estimation  of  hydro- 
chloric acid,  576;  chondroitin  sul- 
phuric acid,  431,  542,  647;  pigments  of 
muscles,  454;  urinary  nitrogen,  548, 
554;  estimation  of  urea,  561;  fatty 
acids  in  the  urine,  608;  nubecula,  615; 
acetanilide,  634;  proteid  in  the  urine, 
640;  nucleoalbumin  in  the  urine,  646, 
647;  melanins,  651,  688;  bile-acids  in 
the  urine,  652. 

Mohr,  Fr.,  reagent  for  hydrochloric  acid, 
374;  chlorine  titration,  617 

Mohr,  L.,  sugar  formation,  306;  urinary 
purines,  579;    oxalic  acid,  583 

Mohr,  P.,  73 

Moitessier,  J.,  blood  catalases,  20;  carbon 
monoxide  metha!moglobin,  207;  chlor- 
ine compounds  in  the  urine,  616; 
Bence-Jones  proteid,  645 

Moleschott,  J.,  764 

Molisch,  H.,  117 

Moll,  L.,  46 

Monari,  A.,  creatinine,  455,  470;  muscle- 
work,  468,  470;    xanthocreatinine,  567 

Monery,  A.,  431 

Montuori,  A.,  297 

Moor,  Ovid,  562 

Moore,  B.,  bile  and  fatty  acids,  398,  422; 
reaction  of  intestine,  407;  synthesis  of 
fat,  421;    fat  emulsion,  422 

Moore,  J.,  115 

Moraczewski,  W.,  formation  of  fjeces,  409; 
digestion  of  casein,  521 ;  diabetes,  593 

Morat,  J.,  work  and  carbohydrates,  468 

Morawitz,  P.,  blood  coagulation,  227,  231, 


232,234;  cadaver-blood,  235;  detection 
of  proteoses,  644 

Morax,  V.,  589 

Moreau,  A.,  711 

Moreau,  J.,  129 

Morel,  A.,  fibrinogen,  172,  173;  blood 
lipase,  185;    hsematogen,  503 

Morgen,  A.,  536 

Morgenroth,  J.,  antichymosin,  25,  361 

Mori,  Y.,  foodstuffs,  utilization  of,  418; 
diets,  764 

Moriggia,  A.,  693 

Morishima,  K.,  liver,  281;   lactic  acid,  461 

Moritz,  366 

Moritz,  F.,  transudates,  258;  phlorhizin 
diabetes,  298;  alimentary  glycosuria, 
298 

Moriya,  G.,  lactic  acid,  460 

Morkowin,  N.,63 

Morochowetz,  L.,  430 

Morris,  G.  H.,  fermentation,  10;  isomal- 
tose,  125;   amylolysis,  128 

Moscatelli,  R.,  ascites,  259,  263;  lactic 
acid,  469,  608 

Mosen,  R.,  222 

Mosse,  M.,  pseudochylose  fluid,  262;  blood 
sugar,  296;  acid  formation  in  the 
stomach,  364;  ethereal  sulphuric  acids, 
589 

Mott,  F.  W.,  cerebrospinal  fluid,  264,  488; 
diseases  of  the  nervous  system,  488 

Mouneyrat,  A.,  102 

Mlihle,  P.,  57 

Muhsam,  J.,  241 

Miiller,  Erich,  digestion  of  cellulose,  397 

Miiller,  Ernst,  bile-acids,  312,  313,  316,  318 

Miiller,  Franz,  high  altitudes,  246,  762 

Midler,  Friedrich,  autolysis  of  pneumonic 
infiltrations,  23,  268,  713;  glucosamine 
from  protein  substances,  32,  66-68,  500; 
mucous-membrane  mucin,  66;  star- 
vation (indican),  404;  fat  absorption, 
424,  425;  ethereal  sulphuric  acids,  589; 
urobilin,  603,  607;  urinary  sulphur,  611, 
612;  demolition  of  aniline,  633;  acetone 
bodies,  668;   faeces  nitrogen,  718 

Miiller,  Johannes,  work  and  sugar  con- 
sumption, 469;  enzymes  of  the  egg- 
yolk,  502 

Miiller,  Julius,  591 

Miiller,  M.,  457 

Miiller,  Paul,  proteoses,  60;  koprosterin, 
337;  excrements,  409;  utilization  of 
casein,  530,  719 

Miifler,  Paul  Th.,  fibrinogen,  172,  188; 
bone-marrow,  172,  437 

Miiller,  W.,  cerebrin,  483,  485 

Miintz,  A.,  539 

Miinzer,  E.,  formation  of  urea,  551;  liver 
and  urinary  nitrogen,  554 

Miither,  A.,  113 

Muirhead,  A.,  552 

Mulder,  G.  J.,  73 

Munk,  H.,  276 

Munk,    I.,    chyle    and    lymph,    251-253; 


IXDEX  TO  AUTHORS. 


833 


sulphocyanides,  343,  611;  contents  of 
the  intestine,  407;  absorption  of  pro- 
tein, 414:  of  sugar,  420;  of  fat,  423,  424, 
427;  fat  synthesis  and  fat  formation, 
421,  442,  445;  smooth  muscles,  47S; 
milk,  52.5;  formation  of  urea,  551; 
elimination  of  phenol,  5S9:  work  and 
catabolism.  471,  620:  reaction  for  bile- 
pigments,  633;  titration  of  sugar.  661; 
starvation,  730,  731;  gelatine,  nutritive 
value  of,  745;  nutritive  value  of  pro- 
teoses, 746;  of  asparagin.  747:  protein 
requirement,  75u;  water  and  metabolism 
7. J  3 

Murray,  Fr.  W.,  3S7 

MuscuJus,  F.,  amylolysis,  12S,  344,  388; 
urochloralic  acid,  632;    urease,  677 

Mygge,  J.,  646 

Mylius,  F.,  iodide  of  starch,  127:  iodo- 
cholalic  acid,  316;  bile  acids,  311,  312, 
315-317 

V.  Naegeh,  C,  126 

Naegeli,  O.,  545 

Nagano,  J.,  intestinal  juice,  378;  absorp- 
tion, 419,  420 

Nagel,  W..  697 

Nakaseko,  R.,  290 

Nakayama,  ^M..  erepsin,  153,  380;  reaction 
for  bile-pigments,  653 

V.  Name,  W.  G..  gelatine,  77,  80 

Nasse,  H.,  blood, 243:  hinph,  254;  spleen, 
273 

Nasse,  O.,  activation  of  oxygen,  5;  pro- 
tein bodies,  27,  42,  79;  dextrines,  129; 
glycolysis,  185:  glycogen,  287,  289,  467, 
46S:  saliva,  344.  346:  musculin,  450, 
452;   smooth  muscles,  477 

Naunyn,  B.,  formation  of  glycogen,  291; 
liver  and  bile,  331-333:  demolition  of 
aromatic  substances,  633 

Nawratzki,  264 

Nebelthau,  E.,  glycogen,  291 ;  hcematopor- 
phvrin,  650 

Neilson,  H.,  342 

Keimarm,  W.,  conjugated  glucuronic  acids, 
122,  123,  609.  efO;  carbohydrats-like 
substance  of  the  liver,  285,  296 

Nencki.  L.,  635 

V.  Nencki,  M.,  oxidation,  8;  protein  sul- 
phur, 28;  putrefaction  of  proteins,  30; 
trvtophane,  102:  blood-pigments,  197, 
201,  211-213:  phyUocyanin,  214;  uro- 
bilinoids,  603:  haematoporphvrin,  213, 
214,  332;  ammonia,  241.  550,' 624,  625, 
745;  diabetes.  300;  gastric  enzjmes, 
354,  355;  gastric  juice,  354,  364; 
splitting  of  esters,  389:  intestinal  di- 
gestion, 398,  399,  401:  indol.  402:  re- 
action of  the  intestine,  407;  butyric 
acid  fermentation,  445;  urea,  455,  549, 
551,  553;  carbamic  acid,  552;  uro- 
rosein,  601,  651;  acid  amides,  630; 
demolition  of  aromatic  substances,  634, 
635,  637;  urine,  odor  of,  639;  melanins, 
688 


Nerking,  J.,  determination  of  fat,  137; 
glycogen,  289 

Nerast,  W.,  permeability  of  a  membrane, 
142;  fluid  chains,  191 

Nessler,  374 

Neubauer,  C,  creatine,  456;  creatinine, 
563,  586,  567;   ammonia,  624 

Neubaner,  O.,  protein  reaction,  43;  alcap- 
tonuria,  598;  urobilinogen,  605;  con- 
jugation of  glucuronic  acid,  632; 
Ehrlich"s  reaction,  675 

Neuberg,  C,  acetone,  32,  668;  glucosa- 
mine, 67,  121,  500,  504;  amyloid,  69, 
70;  amino-acids,  isolation  of,' 92;  cys- 
tine, 92-94;  isoserine,  96;  oxj-amino- 
succinic  acid,  96;  tetraoxyaminocap- 
roic  acid,  96,  431;  diamines,  97,  98 
mannoses,  108;  pentoses,  109,  111-113 
osazones,  117;  levulose.  118,  119,  259. 
glucuronic  acid,  121-123;  conjugated 
glucuronic  acids,  122, 123, 609, 610;  l^'sine 
in  the  blood,  186:  liver,  284;  glycogen, 
290;  deamidation,  305;  cholesterin, 
335,  336:  chondrosin.  431;  galactosa- 
zone,  524;  heteroxanthine,  o79;  esti- 
mation of  phenol,  591;  skatox>i  glu- 
curonic acid,  595;  mineral  metabolism. 
616,  626,  719.  737;  phenylhydrazine 
test,  658;  detection  of  glucuronic  acids. 
667 

Neumann,  -\lb..  orcin  test.  112;  nucleic 
acids,  152-155;  pyrimidine  bases,  152, 
165:  urinarj-  iron,  626:  phenylhy- 
drazine test.  658 

Neumann.  E.,  332 

Neumann,  O.,  nutritive  value  of  alcohol. 
755;  protein  requirement,  766 

Neumann.  R..  754 

Neumann.  Walt.,  57 

Neumeister.  R..  proteoses  and  peptones. 
51.  52.  keratins,  73:  tryptopliane,  102; 
dextrins,  129:  glycogen.  289:  absorp- 
tion of  proteins,  412,  413;  ovomucoid, 
508 

Neusser.  E.,  649 

Nickles,  J.,  509 

Niemilowicz,  L.,  purine  bases,  582;  re- 
ducing power  of  the  urine,  609 

Nierenstein,  E.,  357 

Nilson,  G.,  127 

Nilson,  L.  F.,  milk-fat,  527,  528 

Le  Nobel,  C,  urobilinoids,  214,  603; 
hiematoporphyrin,  649;  acetone  test, 
67 

Noeggerath,  C.  T.,  739 

Noet-Paton,  D.,  h-mph,  253;  liver,  282-. 
formation  of  fa't,  294,  296;  bile,  307, 
328:    work  and  protein,  471 

Noguchi,  H.,  337 

Nolf,  P.,  fibrinogen,  172,  175;  fibrinolysis, 
175:  proteoses  in  the  blood,  ?.83,  415; 
blood  coagulation,  234;  saliva,  340; 
carbamic  acid,  563 

Noll,  A.,  nucleic  acid,  154;  protagon,  488 

V.  Noorden,  C,  spectrophotometry',  218; 


831 


INDEX  TO  AUTHORS. 


alimentary  glycosuria,  298;  diabetes, 
301;  formation  of  sugar,  306;  liver  and 
urinary  nitrogen,  554;  ethereal  sul- 
phuric acids,  5S9;  proteid  in  the  urine, 
040;    metabolism,  733,  7 JO,  752,  753 

Notkin,  J.,  276 

Nothwang,  Fr.,  734 

Novi,  J.,  pseudoha?moglobin,  203;  iron  of 
the  liver,  285;  of  the  bile,  325;  saliva, 
347 

Novy,  F.,  49 

Nowak,  J.,  meat,  476;  respiration  ap- 
paratus, 712;   nitrogen  deficeti,  718 

Nussbaum,  M.,  alveolar  air,  70S;  carbon- 
dioxide  tension,  709 

Nuttal,  G.,  405 

Nylander,  E.,  sugar  test,  116,  655 

Nylen,  S.,  345 

Obermayer,  Fr.,  precipitation  of  protein, 
45;   indican  test,  594 

ObermiiUer,  K.,  saponification,  136;  chol- 
esterin,  334,  335,  336 

Oddi,  R.,  bile  in  the  stomach,  398;  amy- 
loid, 431 ;   acidity  of  the  urine,  544 

Odenius,  R.,  514 

Oertel.  diet  cures,  770,  771 

Oertel,  Horst,  phosphorus  compounds  in 
the  urine,  614;  phosphorus  metabolism, 
719 

Oertmann,  E.,  oxidations,  3,  723 

Oerum,  H.  P.  jr.,  blood-pigments,  217; 
human  bile,  327;  estimation  of  indican, 
595 

Oerum,  H.  P.,  cystic  fluids,  502;  nutritive 
value  of  gelatine,  745 

Offer,  Th.  R.,  glycogen,  294;  nutritive 
value  of  alcohol,  755 

Ofner,  R.  119 

Ogata,  M.,  369 

Ogden,  H.,  598 

Oidtmann,  H.,  lymph-glands,  269;  thy- 
mus, 272;  spleen,  274;  thyroid,  275; 
salivary  gland,  339;  pancreas,  382; 
kidneys,  542;    lungs,  714 

Oker-Blom,  M.,  dissociation,  190;  supra- 
renal capsule,  277 

Okunew,  W.,  56 

Oliver,  G.,  278 

Ollendorff,  G.,  107 

Olsavszky,  V.,  620 

Omeliansky,  23,  397 

van  Oondt,  298 

Oppenheim,  M.,20 

Oppenheimer,  C.,  ferments,  12,  13.  390; 
seralbumin,  181 ;  proteoses  in  the  blood, 
183;  parental  protein  assimilation,  412; 
law  of  surface,  757 

Orban,  R.,  379 

Orgler,  A.,  acetone,  32,  668;  tetraoxy- 
aminocaproic  acid,  96,  431;  uric  acid 
and  nutrition,  569 

Orglmeister,  G.,  97 

Orndorff,  W.  R..  bilirubin,  320,  321 

Orton,  K.  J.  P.,  599,  600 

Osborne,  T.  B.,  proteins,  27,  28,  46,  62; 


nucleic  acids,  152,  156;  pyrimidine 
bases,  152,  156;  ovovitellin,  503; 
ovomucin,  506;    ovalbumins,  507 

Osborne,  W.  A.,  459 

Ost,  H.,  125 

Ostwald,  W.,  catalysis,  14;  action  of  ions, 
168 

Oswald,  A.,  halogen  proteins,  31;  thyroid 
gland,  275-277;    urinary  globulins,  643 

Otori,  J.,  mucin,  67;  transudates,  259; 
autodigestion  of  the  pancreas,  394; 
pseudomucin,  501 

V.  Ott,  415 

Ott,  A.,  transudates,  259;  phosphates, 
619 

Otto,  J.  G.,  blood-sugar,  184,  237;  blood, 
242,  243,  419;  blood-plasma,  quantity, 
238;  blood-pigments,  197,  204,  205, 
218;  skatoxyl-glucuronic  acid,  595; 
estimation  of  sugar,   660,  662 

Overton,  E.,  boundary  layer  of  the  pro- 
toplasm, 142,  143,148;  permeability  of 
the  blood  corpuscles,  195;  of  the 
muscles,  465;  mineral  bodies  of  the 
muscles,  464 

Owen-Rees,  251 

Paal,  C.,  proteins,  27,  49,  51,  53,  59;  gela- 
tine peptone,  79,  SO;    animo-acids,  102 

Pachon,  V.,  coagulation  of  the  blood,  234, 
235;  extirpation  of  the  stomach,  369, 
371;     trypsinogen,    385 

Paderi,  185 

Pages,  C,  coagulation  of  the  blood,  229, 
230;  rennet  coagulation,  520;  milk,  536 

Paijkull,  L.,  exudates,  257,  258,  261,  262; 
bile-mucus,  310 

Painter,  H.  M.,  proteoses,  52;   saliva,  346 

Panek,  K.,  oxyproteic  acids  in  the  mine, 
612,  613;  uroferric  acid,  614;  phos- 
phaturia,  621 

Panella,  A.,  phosphoric  acid,  l86,  457,  477 

Panormoff,  A.,  muscle-sugar,  459;  oval- 
bumin, 507;    egg-white,  508 

Pantanelli,  E.,  87 

Panum,  P.  L.,  serum  casein,  178;  blood  in 
starvation,  244,  732;  transfusion,  245, 
248;  elimination  of  urea,  744;  nutritive 
value  of  gelatine,  745 

Panzer,  Th.,  halogen  protein,  32;  chyle, 
251;  cerebrospinal  fluid,  264;  colloid, 
499,  500 

Paraschtschuk,  S.,  538 

Parastschuk,  S.  W.,  362 

Parens,  E.,  cerebrin,  483;  homocerebrin, 
483 

Parke,  J.  L.,  505,  506 

Parker,  W.  H.,  422 

Parmentier,  E.,  530 

Partridge,  C.  L.,  purine-base  enzymes, 
158,  274,  571 

Pascheles,  W.,  631 

Paschutin,  V.,  lymph,  254;  intestinal 
juice,  378 

Pascucci,  O.,  blood-corpuscles,  193,  194 

Pa.squalis,  G.,  608 


INDEX   TO  AUTHORS. 


835 


Pasteur,  L.,  fermentation,  10,  11;  micro- 
organisms and  digestion,  40o 

Patten,  A.  J.,  cysteine,  28,  94;  histidine, 
lOU;    hexone  bases,  100 

Paul,  Th.,  action  of  poison,  168  uric  acid, 
575 

Pauli,  W.,  protein,  39,  40;  gelatine 
solutions,  78 

Paiily,  H.,  histidine,  99;  adrenaline,  278 

Pautz,  W.,  aqueous  humor,  265;  lactase, 
379;   vitreous  humor,  491 

Pavy,  F.  W.,  carbohydrate  in  proteins,  32, 
293;  isomaltose,  184;  glycogen,  290, 
295;  blood  sugar  and  diabetes,  295-298; 
autodigestion  of  stomach,  372;  work 
and  metabolism,  471;  estim^ation  of 
sugar,  660,  663 

Pawlow,  J.  P.,  biliary  fistula,  307;  saliva, 
342,  348;  stomach  and  gastric  juice, 
349,  351,  353,  356;  gastric  enzymes, 
354,  362;  pylorus-reflex,  368;  pan- 
creatic juice,  382-384,  386;  entero- 
kinase,  383;  pancreas  enzymes,  386, 
389,  392,  397;  ammonia  in  the  blood, 
551;  Fck's  fistula,  552;  urea  formation, 
553 

Paver,  A.,  243 

Peiper,  G.,  223 

Peju,  P.,  173 

Pekelharing,  C.  A.,  trj'ptophane,  54; 
fibrin  ferment  and  blood  coagulation, 
175,  176,  229,  230;  nucleoproteids, 
178.  451 ;  gastric  enzymes,  355,  356,  362 

Pemsel,  W.,  38 

Penny,  E.,  589 

Penzoldt,  Fr.,  acetone,  671,  673 

Pernossi,  L.,  12 

Pernou,  M.,  274 

Peskind,  S.,  193 

Petersen,  P.,  475 

Petit,  A.,  616 

Petrone,  A.,  227 

Petrowski,  D.,  486 

Petry,  E.,  blood-corpuscles,  196;  rennet 
coagulation,  521 

V.  Pettenkofer,  M.,  test  for  bile  acids,  311; 
formation  of  fat,  442-444;  work  and 
metabolism,  471-473,  759;  respiration 
apparatus,  712,  722;  metabolism  ex- 
periments, 716,  718,  741,  759;  diets,  764 

Pfaff,  F.,  307 

Pfannenstiel,  J.,499 

Pfaundler,  M.,  digestion  products,  53,  55; 
nitrogen  in  the  urine,  549 

Pfeiffer,  E.,  woman's  milk,  531,  533 

Pfeiffer,  L.,  688 

Pfeiffer,  Th.,  587 

Pfeiffer,  W.,  574 

Pfleiderer,  R.,  359 

Pfliiger,  E.,  oxidations,  3.  4,  723;  ethereal 
sulphuric  acids,  280:  glvrogen.  288-292, 
434;  diabetes,  301-303.  306:  gases  of 
the  saliva,  340;  bile  and  fattv  acids. 
397,  423;  fat  absorption,  422.  423:  fat 
formation,  443,  445,  741;    nitrogen  in 


meat,    444;     muscle   metabolism,    467, 
469,  471,  473,  474;    ovaries,  498;   gases 
of   milk.    528;     urinarj'   nitrogen,   548; 
estimation  of,  560,  561;    estimation  of 
urea,  557-561,   613;    sugar   tests,   657, 
663,  664;    blood-gases  and  respiration, 
251,  696,  697,  700.  702,  708-711;    res- 
piration apparatus,  712;    N  :  C  quotient 
in   the   urine,    720;     calorific   value   of 
oxygen,  723;    protein  metabolism,  739, 
741,  742,  751;    nutritive  requirements, 
742;    external  temperature  ami  meta- 
bolism, 761 ;    protein  requirements,  765 
Phisalix,  C,  693 
Picard.  J.,  491 
Piccard,  63 
Piccolo,  G.,  hsematoidin  and  lutein,  216, 

498 
Pick,  A.,  358 

Pick,  E.  P.,  proteoses  and  peptones,  57, 
55-57,  60;  serglobuUn,  178;  peptozyme. 
234 
Pick,  Friedel,  295,  296 
Pickardt,  M.,  transudates,  259;   cartilage 

434 
Pickering,  J.  W.,  products  similar  to  the 

proteins,  34,  234 
Piegand,  J.,  259 
Pierallini,  G.,  630 

Piettre,  M.,  blood-pigments,  202,  212 
Piloty,  O.,  glucosamine,  121;    conjugated 

glucuronic  acids.  610 
Pilzecker,  A.,  329 

Pinkus,  S.  N.,  proteins,  30,  39;    crystalli- 
zation, 182.  507,  .508 
Piontkowski,  L.  F.,  351 
Piria,  90 
Planer,  J.,  372 
Plattner,  E.,  310 
Plant.  M.,  614 
Playfair,  764 

Plimmer,  R.  H.,  lactase,  386 
Plosz,  P.,  blood  corpuscles,  194;  liver,  281, 
282:  elimination  of  proteo.ses.  414; 
nutritive  value  of  proteoses,  746:  uri- 
nary pigments,  601;  urinary  protein, 
640 
Poda.  H.,  excrements,  409;   absorption  of 

casein.  719 
Podiischka,  P..  574 
Poelil.    A.,    intestinal    putrefaction,    405; 

spermine,  496 
Pohl,    J.,    oxidations.    8;     dextrin,    129; 
estimation  of  globulin,  180,  646;    liver, 
281;     urea   formation,    551;     allantoin, 
574.  584;    oxalic  acid.  630;    demoliiion 
of  fatty  bodies.  630 
Poleck.  egg.  505,  509 
Polimanti.    O.,    estimation    of    fat,    137; 

fattv  degeneration,  443 
Politis.  G.,  747 
Pollak.  L.,  glutinase,  391,  392;   sulphocy- 

anide,  631 
Pollitzer,  S.,  746 
Pommerehne,  H.,  563 


836 


INDEX  TO  AUTHORS. 


Ponfick,  E.,  248 

Ponomarew,  Brunner's  glands,  377 
Popel,W.,244 

Popielski,  L.,  enterokinase,  383;   pancrea- 
tic juice,  383,  384 
Popoff,  X.,  415 
Porcher,  Ch.,  lactose,  539;    urinary  indi- 

can,  592-595 ;   skatol  red  and  urorosein, 

596;   uroerythyrin,  607 
Porges,  O.,  179 
Porteret,  E.,  291 
Portier,  P.,  fermentation,  21;    lactose  in 

the  intestine,  419 
Tosner,  C,  semen,  495,  496;    urinary  pro- 
tein, 640 
Posner,  E.  R.,  mucin,  67,  68;    mucoids, 

360;  protagon,  482 
Posselt,  L.,  82 
Posternak,   S.,   musculamine,   457;    oxj''- 

methylphosphoric  acid,  458 
Pottevin,  H.,  isomaltose,  125;    syntheses 

of  esters,  390 
Pouchet,   A.   G.,   carmine,   456;     urinary 

purines,    578;      urinary    poison,     615; 

lungs,  714 
Poulet,  v.,  713 
Poulsson,  E.,  455 

Pozerski,  E.,  kinases,  383;  secretin,  384 
Pozzi-Escot,  E.,  19 
Prausnitz,  W.,  phlorhizin  diabetes,   298; 

excrements,  409;    absorption  of  casein, 

719;  metabolism  in  starvation,  729,  730 
Predteschensky,  E.,  761 
Pregl,    Fr.,   protein   hydrolysis,   88,    101; 

carbon  monoxide  ha'mochromogen,  209; 

dehydrocholon,    311,    316;     bile-acids, 

316-318;     intestinal    juice,    377,    378; 

ovalbumin,  507;    oxj^^roteic  acid,  613; 

peptides    in    the    urine-,    614;     C.  :  N. 

quotient,  628 
Presch,  W.,  urinary  sulphur,  611;    hypo- 

sulphrites,  612 
Preusse,   C,   phenols  in  the  urine,   591; 

behavior  of  aromatic   substances,  633, 

639 
Prevost,  J.   L.,  bile,   426;    formation  of 

urea,  553 
Preyer,  W.,  globin,  208;    blood-crystals, 

208;  placenta-pigment,  512 
Preysz,  K.,  620 
Pribram,  E.,  633 
Pribram    R.,  623 
Pribram,  H.,  453 
Prochownick,  L.,  513 
Proscher,    F.,   bilirubin,   321;    milk,   529, 

536;    Erhlich's  urine  test,  675 
Prutz,  W..  592 
Prynn,  O.,  spleen  and  digestion,  274,  385; 

trypsin,  383 
Pugiie.se,  A.,  232 
Puis,  J.,  525 
Pupkin,  Z.,  223 

Quevenne,  Th.,  lymph,  252;  witch's  milk, 
534 


Quincke,  G.,  517 

Quincke,  H.,  hsematoidin,  216;  iron 
preparations,   245;     poikilozytosis,   247 

Quinquaud,  Ch.,  urea,  240,  242;  muscle- 
work,  468;   fatty  acids  in  the  urine,  629 

Raaschou,  C.  A.,  guanylic  acid,  155,  156 
Rachford,  B.  K.,  pancreatic  diastase,  388; 

trypsin  digestion,  393;   bile  and  fat,  398 
Radenhausen,  P.,  517 
Radziejewski,  S.,  442 
Radzikowski,  C,  350 
Raehlmann,  E.,  491 
Raikow,  P.  N.,  28 
Raineri,  G.,  512 
Ramsden,  W.,  61 
Ranke,  H.,  569 
Ranke,  J.,  division  of  blood,  249;    bile, 

307 
Ransom,  H.,  haemolysis,  193,  337 
Rapp,  R.,  10 

Rauchwerger,  D.,  cholesterin,  335,  336 
Raudnitz,  R.  W.,  milk  and  casein,  515, 

519,  521 
Reach,  F.,  tyrosine,  89;  pseudopepsin,355; 

digestion,  370;   protein  absorption,  413; 

muscular  activity,  474 
Reale,    E.,    oxalic    acid,    582;     urinary 

sulphur,  611 
Reese,  H.,  614 
Regnault,   H.   V.,   cutaneous  respiration, 

695;   method  of  respiration  experiment, 

712;  nitrogen  deficit,  722 
Reh,  A.,  269 
Reich,  O.,  625 
Reichel,  H.,  521 
Reich-Herzberge,  F.,  395 
Reinbold,    R.,    Molisch's    reaction,    117; 

met  haemoglobin,  204;  trypsin  digestion, 

394;  benzoylation  of  carbohydrates,  659 
Reinders,  W.,  14 
Reinecke,  441 
Reiset,    J.,    cutaneous    respiration,    695; 

method  of  respiration  experiment,  712, 

722;   nitrogen  deficit,  718 
Reiss,  E.,  180 
Reissner,  O.,  376 
Reitzenstein,  A.,  578 
Rekowski,  L.,  637 
Rennie,  J.,  pancreas,  302,  381 
Renwall,  G.,  625 
Rettger,  L.  F.,  385 
Reuss,  A.,  transudates,  259,  261 
Reye,  W.,  174 
Reynolds,  J.  E.,  671 
de  Rey-Pailhade,  J.,  enzymes,  8,  19 
V.  Rohrer.  L.,  acid  compounds  of  proteins, 

38;   acidity  of  the  urine,  545 
Riazantseff,  N.  V.,  elimination  of  nitrogen, 

745,  762 
Ribaut,  H.,  enzyme  actions,  17,  19 
Richards,  A.  N.,  albuminoids,  75,  76,  77; 

hexone  bases,  100;   saliva,  343 
Richet,     Ch.,    gastric    juice,     349,    353; 

digestion,    368;     fat    formation,    446, 


INDEX  TO  AUTHORS. 


837 


urea,  550;  uric  acid,  574;  thalassin, 
693;  method  of  respiration  experiment, 
713,  722;   law  of  surface,  756 

Richter,  Max,  496 

Richter,  P.  F.,  liver,  186,  284,  554 

Riecke,  R..  587 

Rieder,  H.,  718 

Riegel.  M.,  518 

Riesel,  secretion  of  gastric  juice,  353 

Riegler,  R.,  210 

Riess,  L.,  oxymandelic  acid,  597;  lactic 
acid,  608 

Ringer,  L.,  229 

Ringstedt,  O.  T.,  544 

Ritter,  A.,  plilorhizen  diabetes,  298;  fat 
absorption,  42;  fermentation  of  urine, 
677 

Ritter,  E.,  bile,  328,  329:   gall-stones,  334 

Ritter,  E.,   cholesterin,  338 

Ritthausen,  H.,  proteins,  29,  38;  leucin- 
imide,  87;    milk,  525 

Riva,  A.,  urinary  pigments,  601,  607,  650 

Rivalta,  F.,  258 

Roberts,  W.,  pancreas  rennet,  396;  esti- 
mation of  protein,  646;  estimation  of 
sugar,  663 

Roch.  G.,  642 

Rockwood,  C.  W.,  614 

Rockwood,  D.,  bile  and  fatty  acids,  398, 
422;  fat  absorption,  422;  reaction  of 
the  intestine,  407 

Rockwood,  E.  W.,  protein  assimilation 
and  absorption,  413;    uric  acid,  570 

Rodier,  A.,  243 

Roden,  H.,  361 

Roeder,  G.,  pyrimidine  bases,   164 

Rolunann,  F.,  oxidation  en2}Tnes,  8,  18; 
leucin  ethylester,  86;  detection  of  glu- 
cose, 118;  amylolysis,  125,  345;  gly- 
colysis 185;  fat  in  the  blood,  241; 
diastase,  185,  251,  295,  297;  glycogen 
formation,  291;  intestinal  juice,  378; 
bile  and  putrefaction,  406:  absorption, 
419,  421,  424;  muscles,  447,  466;  casein 
salts,  519;  phosphorus  metabolism,  619, 
719,  738;  sebum,  691;  anal-gland 
secretion,  692;    artificial  nutrition,  739 

Rohrig,  A.,  metabolism  of  the  muscles, 
467;    cutaneous  respiration,  695 

Rose,  B.,  521 

Rosing,  E.,  6 

Roger,  G.  H.,  280 

Rohde,  E.,  43 

Rokitausky,  P.,  608 

Rona,  P.,  histone,  62;  gelatine,  78;  <X- 
proline,  101;  sugar  formation,  296; 
duodenal  secretion,  377;  protein  re- 
generation, 417 

Ronchi,  J.,  cutaneous    respiration,    695 

Roos,  E.,  iodothyrine,  276;  phosphorus 
metabolism,  619;  sugar  tests,  658, 
659 

Roosen,  O.,  568 

Rosa,  E.,  713 

Rosemann,  Rud,  milk,  539;    eliminatio 


of  nitrogen,  744;  nutritive  value  of 
alcohol,  755 

Rosenbach,  (),  urine  tests,  652,  675 

Rosenbaum,  A.,  blood  catalases,  20; 
glycolys  s,  303 

Rosenberg,  Br.,  298 

Rosenberg,  S.,  secretion  of  bile,  308; 
pancreatic  juice,  383;  bile  and  putre- 
faction, 406;  pancreas  and  absorption, 
418,  421,  425 

Rosenfeld,  Fritz,  indican,  593;  volatile 
fatty  acids,  608 

Rosenfeld,  G.,  fat  and  fat  formation,  282, 
283,  442,  443;  glycogen  and  fat,  283; 
uric  acid,  569;  phenylhydrazine  test, 
658;    acetone  bodies,  669 

Rosenfeld,  M.,  210 

Rosenfeld,  R.,  184 

Rosenheim,  Th.,  750 

Rosenqvist,  E.,  306 

Rosenstein,  A.,  chvle  and  Ivmph  absorp- 
tion, 252,  253,  414,  420.  423 

Rosenstein,  W.,  299 

Rosenthal,  J.,  712 

Rosin,  H.,  levulose,  119,  665;  indican, 
594;  skatol  pigments,  596:  reducting 
power  of  the  urine,  609;  Rosenbach's 
urine  test,  675 

Rossi,  O.,  264 

Rost,  E.,  gallic  and  tannic  acids,  637; 
salts  and  metabolism,  754 

Rostoski,  O.,  645 

Roth,  W.,  256 

Rothberger,  C.  J.,  liver,  280;  carbamic 
acid,    552 

Rothera,  C.  H.,  protein  nitrogen,  27; 
cystine,  92 

Rotmann,  F.,  259 

Rotschv,  A.,  214 

Roux.  E.,  126 

Rovida,  C.  L.,  hyaline  substance,  141,  194, 
267 

Rovighi,  A.,  putrefaction,  405,  589 

Rowland,  S.,  fermentation,  10;  leinases, 
273;  muscle  enzjTnes,  454 

Rubbrecht,R.,lS8 

Rubner,  M.,  protein  sulphur,  28;  sugar 
reaction,  117,  658,  665;  utilization  of 
foodstuffs,  417,  421,  424,  530,  727,  763; 
nitrogen  in  meat,  444;  fat  formation, 
445;  milk,  532;  nitrogen  of  fsces,  718; 
heat  of  combustion  of  foodstuffs,  724- 
727;  metabolism  experiments,  716,  726, 
730,  752,  756-758,  761;  law  of  surface, 
757,  758 

Rubow,  W.,  464 

Riidel.  G.,  575 

Ruff,  O.,  107 

Ruge,  E.,  404 

Rulot,  H.,  fibrinolysis,  174,  175 

Rumpf,  Th.,  sugar  formation,  306;  esti- 
mation of  phenol,  589;    ammonia,  625 

Runneberg,  J.  W.,  261 

Ruppel,  W.  G.,  protagon.  481,  482;  milk- 
fat,  530;  vernix  caseosa,  691 


■838 


INDEX  TO   AUTHORS. 


Russel,  523 
Russo,  M.,  687 

Rywosch,  D.,  glycolysis,  185;  blood- 
corpuscles,  193 

iSaarbach,  L.,  204 

Sabanejew,  A.,  60 

Sabbatani,  S.,  229 

Sacharjin,  238 

Sacharow,  N.,  57 

Sachs,  Fritz,  nucleases,  153,  380,  395; 
elimination  of  salts,  364 

Sachsse,  R.,  carbohydrate,  116,  127 

Sackur,  O.,  casein,  519,  520 

Sadikoff,  W.,  77 

Sahli,  H.,  hsemometer,  219;  titration  of 
sugar,  663 

Saiki,  T.,  608 

Saillet,  urobilin  and  urobilinogen,  601- 
606;    hfpmatoporphyrin,  213,  650 

de  Saint  Martin,  L.,  205 

Saint  Pierre,  C,  711 

Saito,  S.,  lactic  acid,  241,  461 

Salaskin,  S.,  digestion  products,  53; 
plastein,  56;  leucinimide,  87;  alkalinity 
of  the  blood,  223;  anunonia,  241,  551, 
624,  745;  erepsin,  380;  urea,  550,  553, 
561 ;  liver  and  acid  formation,  554,  572; 
uric  acid  formation.  572 

Salkowski,  E. ,  oxidation  enzymes,  8 ;  auto- 
digestion,  22;  putrefactive  products, 
30,401;  protein,  40,  43;  pseudonucleic 
acid,  46,  521,  522;  pseudonuclein,  47; 
proteoses,  52,  644;  skatolcarbonic  acid, 
102,  596;  pentoses,  110-112,  290,  666; 
glucuronic  acid,  121;  cerebrospinal 
fluid,  264;  synovin,  265;  liver  proteid 
282;  glycogen,  290;  cholesterin,  336; 
saliva,  347;  pancreas,  391;  indol,  403; 
adipocere,  443;  nitrogen  of  meat,  476! 
ovomucoid,  508;  casein,  521,  522; 
xirea,  550,  551,  553;  creatinine,  565, 
566;  uric  acid,  573,  576;  purine  bases, 
581;  oxalic  acid,  582,  583,  630; 
allantoin,  584;  hippuric  acid,  585; 
phenaceturic  acid,  588;  ethereal  sul- 
phuric acid,  589;  indican,  595;  urobilin, 
605,  644;  urine,  fatty  acids  of,  608,  677; 
carbohydrates  of,  609;  sulphur  com- 
pounds of,  611,  612;  alkalies  of,  623; 
estimation  of  the  sulphur  acids  of  the 
urine,  623;  destruction  of  various  sub- 
stances, 631,  632,  634,  636;  h^mato- 
porphyrin,  649,  650;  fermentation  test, 
657;  estimation  of  acetone,  673;  water 
and  metabolism,  753 

Salkowski,  H.,  putrefaction,  401,  585; 
demolition  of  aromatic  substances,  634 

Salomon,  George,  glycogen,  148;  purine 
bases,  158;  lactic  acid,  241;  urme 
purines,  578-580 

Salomon,  H.,  dieamidation,  305;  oxalic 
acid,  583;   acetone,  670 

Salomon,  W.,  551 

Salvioli,  G.,  414,  415 


Sammis,  J.  L.,  767 

Samuely,  F.,  melanoidins,  30,  689;  amino- 
acids  in  the  urine,  614;  cystine,  demoli- 
tion of,  631 

Sanders-Ezn,  H.,  761 

Sandmeyer,  W.,  pancreas  diabetes,  301; 
absorption,  418,  425,  426;  phosphorus 
metabolism,  719 

Satta,  G.,  urinary  nitrogen,  549;  acetone 
bodies,  669 

Sauerbeck,  E.,  pancreas,  302,  381 

Sawitsch,  W.,  385 

Sawjalow,  W.,  plastein,  56;  chymosin,  362 

Schafer,  E.,  coagulation  of  blood,  229; 
suprarenal  capsule,  287 

Schaffer,  Ph.,  uric  acid,  578;  ammonia,  625 

Schaffer,  F.,  28 

Schalfejeff,  M..  hsemin,  211,  212 

Schardinger,  F.,  460 

Scheermesser,  \V.,  peptons,  57,  58,  80 

Scheibe,  A.,  532 

Scheibler,  C,  124 

Schemiakine,  A.  J.,  365 

Schenck,  Fr.,  detection  of  glucose,  118; 
blood-sugar,  240,  297,  469 

Schenck,  M.,  protein  substances,  oxida- 
tion of,  32,  582;    martamic  acid,  154 

Schepowainikow,  N.  P.,  383 

Schepski,  N.  W.,  745 

Scherer,  J.,  lymph,  252;  inosite,  458,  459, 
meta-  and  paralbumin,  500 

Scheuer,  M.,  353 

Scheunert,  A.,  367 

Schierbeck,  N.  P.,  saliva,  345;  gases  of 
the  stomach,  372;  tryj^sin  digestion, 
393;   faeces,  408 

Schiff,  A.,  357 

Schiff,  H.,  protein,  27;  biuret  reaction,  43; 
cholesterin,  336;  urea,  555;  uric  acid, 
576 

Schiff,  M.,  spleen,  274;  liver,  280;  liver 
sugar,  295;  bile,  309,  426;  charging 
theory,  364,  385 

Schindler,  S.,  271 

Schittenhelm,  A.,  xanthine  oxidases,  18, 
158,  273,  571,  574;  elastin,  75;  amino- 
acids, 83-85, 91 ,  614, 630;  a-proline,  101 ; 
nucleic  acids,  153;  purine  bases,  158, 
163,  408;  coagulation  of  the  blood,  231, 
233;  spleen,  273;  uric  acid,  275,  571, 
574;  estimation  of  ammonia,  625; 
cystinuria,  675 

Schlatter,  K.,  digestion,  370,  371 

Schlesinger,  A.,  345 

Schlesinger,  W.,  606 

Schlosing,  Th.,625 

Schlossberger,  J.  E.,  milk,  534,  539 

Schlossmann,  A.,  milk,  526,  529,  532 

Schmey,  M.,  liver,  285;  muscle,  464,  477 

Schmid,  Jul.,  581 

Schmidt,  Ad.,  intestinal  putrefaction,  400; 
excrements,  410 

Schmidt,  Albr.,  497 

Schmidt,  Alex.,  catalysis,  10;  cell  protein, 
141,  228,  271;  coagulation  of  the  blood. 


INDEX   TO  AUTHORS. 


S39 


176,  177,  261,  227-232;  fibrinoplastic 
substance,  178,  221,  228;  blood  cor- 
puscles of  the  frog,  195;  leucocytes, 
221.  227-229;    blood-gases,  698. 

Schmidt,  C,  serum,  188;  blood,  238,  239; 
lymph,  252^  transudates,  257;  buccal 
mucus,  341;  saliva,  346;  gastric  juice, 
353,  354;  pancreatic  juice,  387;  biliary 
fistula,  406;  absorption  of  fat,  424; 
osteomalacia,  439 

Schmidt,  C.  H.  L.,  31 

Schmidt,  C.  W.,  714 

Schmidt,  E.,  455 

Schmidt,  Fr. ,  670 

Schmidt,  P.,  579 

Schmidt-Mulheim,  A.,  coagulation  of  the 
blood,  171;  protein  absorption,  413, 
414,744 

Schmidt-Nielsen,  S.,  enzymes,  12;  chy- 
mosin,  362 

Scluniedeberg,  O.,  oxidations,  7;  protein 
crystals,  38;  desamino-albuminic  acid, 
49;  salmine,  63;  onuphin,  69;  campho- 
glucuronic  acid,  122,  638;  nucleic  acids, 
152,  154,  155;  nucleosin,  165;  fen  at  in, 
282;  chondroitin  sulphuric  acid,  431, 
432;  urea,  551;  hippuric  acid,  586,  587; 
histozym,  588;  phenolgl neuronic  acid, 
590;  indoxylglucuronic  acid,  595;  chi- 
tin,  686;    melanin  substances,  688 

Schmitz,  K.,  putrefaction,  405,  407,  589 

Schneider,  A.,  238 

Schneider,  E.,  saliva,  343;  kynurenic 
acid,  600 

Schneider,  H.,  tyrosinases,  18;  melanins, 
690 

Schoffer,  A.,  85 

Schonbein,  C.  F.,  623 

Schondorff,  B.,  urea,  240,  455,  532;  esti- 
mation of,  562;  thyroidea,  277;  glyco- 
gen, 288,  292,  459;  uric  acid,  569;  pro- 
tein metabolism,  742,  743 

Schone,  A.,  Ill 

Scholz,  H.,  indican,  593 

Shore,  L.  E.,  absorption,  415,  416 

Schotteliug,  M.,  405 

Schotten,  C,  fellic  acid,  318;  intestinal 
putrefaction,  586;  fatty  acids  in  the 
urine,  608,  629;  damaturic  and  damolic 
acids,  616;  behavior  of  aromatic  sub- 
stances, 633,  634 

Schoubenko,  G.,  28 

Schoumow-Simanowski,  E.  O.,  pepsin, 
355;    gastric  juice,  364,  371 

Schreiber,  E.,  571 

Schreiner,  Ph.,  496 

Schreuer,  M.,  nitrogen  of  meat,  477; 
calorific  value  of  nitrogen,  727;  protein- 
overfeeding,  744,  752 

Schrodt,  M.,  437 

V.  Schroeder,  W.,  uric  acid,  240,  241,  547, 
572;     urea,   551,   553 

Schroter,  F.,  153 

Schrotter,  H  ,  proteoses  and  peptone,  52, 
53,  60 


Schryver,  S.  B.,  23 

Schiile,  353 

SchiUe,  A.,  343 

Schutz.,  E.,  digestion  products,  55;  esti- 
mation of  pepsin,  357,  358;  stomach, 
366;    lactic  acid,  608 

Schiitz,  J.,  pepsin,  estimation  of,  357; 
action  of,  359;    bile  and  fat,  398 

Schutze,  .\lb.,  186 

Schiitzenberger,  P.,  proteins,  29,  34,  51,  59 

Schultze,   B.,  445 

Schultze,  E.,  548 

Schultzen,  O.,  diabetes,  300;  urea,  550, 
551;  oxymandelic  acid,  597;  lactic 
acid,  608;  acid  amides,  631;  sarcosin, 
631;  behavior  of  aromatic  substances, 
633,  635 

Schulz,  .\rth.,  215 

Sclmlz,  Fr.  N.,  protein,  28,  31,  33,  38,  41, 
59;  oxyjirotein,  31;  hi.stones,  62,  208; 
galactosamine,  65,  71,  121;  estimation 
of  fat,  137;  seralbumin,  181;  starva- 
tion, 244,  730 

Schulz,  H.,  429 

Schulze,  C,  345 

Schulze,  E.,  hydrolytic  products  of  pro- 
teins, 29,  84,  85,  87,  90,  91;  hexone 
bases,  96,  100;  hemicelluloses,  130; 
isocholesterin,  337;    lecithans,  144 

Schulze,  E.,  522 

Schulze,  F.  E.,  166 

Schumburg,  W.,  chymosin,  361;  metabo- 
lism, 758 

Schumm,  O.,  blood,  217,  247;  spleen.  273; 
sugar  formation,  306;  pancreatic  cysts, 
387 

Schunck,  C.  A.,  pigments,  197,  505 

Schunck.  E.,  601 

Schur,  H.,  uric  acid,  569, 570, 574;  urinary 
purines,  579 

Schurig,  285 

Schuster,  A.,  utilization  of  foodstuffs,  418; 
diets,  770 

Schuurmanns-Stekhoven,  202 

Schwalbe,  E.,  blood  plates,  227;  fat,  283, 
443 

Schwann,  Th.,  biliary  fistula,  307,  406 

Schwarz,  H.,  117 

Schwarz,  Hugo,  elastin,  75,  76,  89 

Schwarz,  L.,  protein  compounds,  49; 
secretion  of  hydrochloric  acid,  364; 
acetone  bodies,  669 

Scliwarz.  O.,  antipepsin,  373;  aceto-acetic 
acid,  672 

Schwarzschild,  M.,  biuret  bases,  35; 
trypsin,  390,  395 

Schweissinger,  O.,  642 

Schwinse,  W.,  243 

Scofield,  H.,  323 

Sebelein,  J.,  peptones,  52;  digestion  of 
casein,  521;  milk,  515,  521,  522,  525, 
528 

Seegen.  J.,  blood-sugar,  118.  184.  295,  296, 
468,  473;  carbohydrate  substance  in  the 
liver,  285,  296,  297;    amylolysis,  344; 


840 


INDEX  TO  AUTHORS. 


respiration,  712;    nitrogen  deficit,  718; 
metabolism  and  water,  753 

Seelig,  P.,  4U5 

Seemann,  J.,  proteins,  oxidation  of,  32,  78; 
carboiiydrate  in  the  proteins,  33,  508; 
nucleic  acid,  154;  erepsin,  380;  in- 
testinal contents,  400;  absorption,  413; 
ovomucoid,  508 

Segale,  M.,  166 

Seitz,  W.,  287 

Selitrenny,  L.,  78 

Seliwanoff,  Th.,  levulose,  119,  665 

Selmi,  24 

Semmer,  G.,  195 

Senator,  H.,  593 

Senkowski,  M.,  bile-acids,  316 

Senter,  G.,  20 

Serdjukow,  A.,  368 

Sertoli,  E.,  701 

Sestini,  L.,  568 

Setschenow,  J.,  blood-gases,  697,  699,  701 

Shepard,  C.  U.,  585 

Shore,  L.  E.,  protein  absorption,  415,  416 

Siau,  R.  L.,  isomaltose,  184;  phlorhizin 
diabetes,  298 

Sieber,  N.,  protein  sulphur,  28;  glycolysis 
185;  blood-pigments,  201,  211,  213 
haematoporphyrin,  213,  214,  215,  332 
urobilinoids,  214,  603;  diabetes,  300 
gastric  juice,  354,  355,  362,  371;  in. 
testinal  digestion,  399;  Umikoff's  re- 
action, 532;  urorosein,  601,  651;  nitro- 
benzaldehyde,  636;    melanins,  688,  689 

Siegert^  F.,  441 

Siegfried,  M.,  hydrolysis  of  proteins,  29; 
peptone  substances  and  kyrines,  54,  58, 
59,  60,  80;  reticulin,  80,  81,  428;  glu- 
tamic acid,  88;  nitrotoluene  sulpho- 
compounds,  92;  lysine,  99;  pseudo- 
haemoglobin,  203;  jecorin,  283;  phos- 
phocarnic  acid,  457,  458,  462,  470,  474; 
orylic  acid,  523;  milk  nucleon,  532; 
carbanimo-acids,  701 

Silbermaim,  M.,  isoserine,  96;  oxyanimo- 
succinic  acid,  96 

Silbermann^  O.,  333 

Simacek,  E.,  fermentation,  21,  396;  lactic 
acid,  461 

Simon,  G.,  milk,  526,  528 

Simon,  O.,  autolysis,  23,  268,  714;  car- 
bohydrate substance  of  the  liver,  296 

de  Sinety,  L.,  665 

Siven,  V.  O.,  uric  acid,  569,  570;  protein 
metabolism,  738,  750,  765 

Siwertzow,  D.,  145 

Sjoqvist,  J.,  proteins,  38,  60;  estimation  of 
hydrochloric  acid,  375,  376;  urinary 
nitrogen,  546,  554;  urea  estimations, 
561 

Skita,  A.,  fibroin  hydrolysis,  82-84,  89 

Skraup,  Zd.,  protein  hydrolysis,  29,  78; 
oxyanimo  acids,  96,  ioi;  caseanic  and 
caseinic  acids,  101;  benzoylation  of 
carbohydrates,  117;   kyrines,  58 

Slosse,  A.,  urinary  nitrogen,  553,  744 


Slowtzoff,  B.,  pentosanes,  111;  liver,  286; 
semen,  495;    metabolism,     57,  759 

Smals,  Fr.,  575 

Smirnow,  A.,  636 

Smith,  F.,  693 

Smith,  Herbert,  saliva,  345,  346;  bones, 
435 

Smith,  Lowain,  chlorosis,  246;  quantity 
of  blood,  248;   oxygen  tension,  707,  708 

Smith,  W.  G.,  654 

Smith,  W.  J.,  urinary  sulphur,  612; 
demolition  of  sulphur  compounds,  631 

Smits,  H.,  46 

Socin,  C.  A.,  293 

Socoloff,  N.  326 

Soldner,  F.,  milk,  518-520,  525-527,  531, 
533-535 

Sorensen,  S.  P.  S.,  amino-acids,  83; 
ornithin,  97;   a-proline,  102 

Soetber,  F.,  621 

Solera,  L.,  343 

Solley,  Fr.,  gelatine,  77,  79 

Sollmann,  T.,  gall-bladder,  329;  muscles, 
449,  453;    fibroma  of  uterus,  502 

Sommer,  A.,  283 

Sommerfeld,  327 

Sonden,  K.,  respiration  aparatus,  713, 
722;    metabolism,  756,  758,  768 

Sorby,  H.  C.,  509 

Sourdat,  534 

Southgate,  393 

Soxhlet,  F.,  glucose,  117;  maltose,  125; 
fat  formation,  445;  milk,  518,  520,  526, 
536,  538;    titration  of  sugar,  661 

Spampani,  G.,  538 

Spangaro,  S.,  170 

Speck,  C,  gas  exchange,  713,  759-762 

Spiegler,  A.,  734 

Spiegler,  E.,  protein  reagent,  642;  mel- 
anins, 689 

Spiro,  K.,  proteins,  38,  45;  phenylalanine, 
78;  glycocoll,  83;  serglobulin,  179; 
blood  coagulation,  231,  232,  234;  pep- 
tozym,  234;  lactic  acid,  469;  rennet 
coagulation,  520,  521 

Spitzer,  W.,  oxidation  enzymes,  8,  18,  20; 
xanthin  oxidases,  18;  glycolysis,  20, 
185,  302;  liver  protein,  282;  uric  acid 
formation,  571 

Spriggs,  E.  J.,  357 

Ssubotin,  M.,  536 

Staal,  J.  Ph.,  596 

Stade,  W.,  363 

Stadelmann,  E.,  tryptophane,  102;  ict- 
erus, 216,  332,  333;  suprarenal  capsule, 
277,  330;  bile,  307-309,  328,  331-333, 
426:  intestinal  putrefaction,  407;  elin  i- 
nation  of  nitrogen,  554;  elimination  of 
ammonia,  624;  peptonuria,  643;  /^-oxy- 
butyric  acid,  673;  blood  in  diabetes,  702 

Stadthagen,  M.,  diamines,  24,  615; 
adenine,  162;  xanthocreatinine,  567; 
urinary  sulphur,  (111;  poison  of  urine, 
615;    cystine,  676 

Staedeler,  G.,  324 


INDEX  TO  AUTHORS. 


841 


Staehelin,  R.,  258 

Stanek,  V.,  145 

Stange,  M.,  132 

Starke,  J.,  proteins,  40,  182;   globvilin,  46 

Starke,  K,  181,  182 

Starling,   E.    H.,   lymph   formation,   255, 

256;    intestinal  enzymes,  379;    entero- 

kinase  and  trypsinogen,  379,  383-386; 

secretin,  384;    pancreatic  erepsin,  391 
Stassano,  H.,  386 
Stavenhagen,  A.,  10 
Steensma,  F.  A.,  402 
Steilf,  R.,  589 
Steigef,  E.,  96 

Steil,  H.,  muscle-fat,  474,  475 
Stein,  G.,  334 
Steinitz,     phosphorus    metabolism,    619, 

719;   C. :  N.  quotient,  628,  720;   lactose 

in  the  urine,  665;    artificial  nutrition, 

739 
V.  Stejskal,  R.,  240 
Sternberg,  S.,  526 
Stepanek,  J.  O.,  502 
Stern,  H.,  331 
Stern,  Heinrich,  496 
Stern,  L.,  20 
Stern,   R.,  bile    329;    ethereal  siilphuric 

acids,  589 
Steudel,    H.,    mucin,    67;     arginine,    97; 

glucosamine,    121;    nucleic  acids,   151; 

pyrimidine,  bases,  152,  164-166 
Stewart,  C.  W.,  359 
Stewart,  G.  N.,  blood,  236;    muscle,  449, 

453 
Steyrer,  A.,  muscle,  453,  472;  urine,  546 
Sticker,  G.,  saliva,  343,  346 
Stiles,  P.,  298 
Stintzing,  R.,  467 
Stockmann,  R.,  471 
StofTregen,  A.,  643 
Stohmann,  F.,  digestion  of  cellulose,  397; 

calorific  determinations,  724 
Stoklasa,  J.,  fermentations,  21,  396,  523; 

lecithin,   146;    glycolysis,   302;     lactic- 
acid  formation,  461 
Stokvis,    B.   J.,   bile-pigments,  322,   323, 

603,  653;    benzoic  acid,  588;    urobilin, 

604,  644;   haematoporphyrin,  649 
Stolnikow,  J.,  646 

Stolte,  K.,  550 

Stoltz,  Fr.,  278 

Stone,  W.  E.,  Ill 

Stookey,   L.   B.,  gelatine,   79;    glycogen, 

292 
Stoop,  F.,  cystine,  93;   serine,  93,  96 
Storch,  v.,  milk,  517,  539 
Stradomsky,  N.,  oxalic  acid,  583,  630 
Strashesko,  N.  D.,  350 
Strassburg,  G.,  gases  of  the  lymph,  251; 

carbon-dioxide  tension,  709,  712 
Strassburger,  J.,  410 
Straub,  W.,  glj'^cosuria,  299;   metabolism, 

734,  754 
Straus,  J.,  262 
Strauss,  Edw.,  spongin,  81,  82 


Strauss,  H.,  le\nilose,  118,  119,  184,  259; 
blood,  123;  transudates,  259,  262;  bile, 
326;  lactic-acid  fermentation,  371; 
urine,  546 

Strecker,  A.,  lecithin,  143;   bile-acids,  315 

Strickler,  E.,  528 

Strohmer,  F.,  445 

Stusiewicz,  B.,  747 

Struve,  H.,  649 

Strzyzowski,  C,  213 

Subbotin,  V.,  blood,  244;  alcohol,  754 

Sugg,  E.,  523 

Suida,  W.,  334 

Sulima,  A.  Th.,  digestion,  370,  400 

O'Sullivan,  C,  13 

Sundl?erg,  C,  356 

Sundvik,  E.,  glucosamine,  121;  xanthine 
bodies,  157;  uric  acid,  568;  conjugated 
glucuronic  acids,  610,  632;  chitin,  686; 
psylla  alcohol,  692 

Suter,  F.,  protein  sulphor,  28;  thiolactic 
acid,  28,  74,  94 

Suto,  K.,  663 

Suzuki,  W.,  92 

Svenson,  N.,  753 

Swain,  R.  E.,  103 

Symmers,  D.,  614 

Syniewski,  V.,  starch,  126;  dextrin,  128 

Szekely,  S.,  137 

V.  Szontagh,  F.,  milk  enzymes,  523;  case- 
ins, 529,  531 

Szydlowski,  Z.,  361 

Szymonowicz,  L.,  278 

Takamine,  J.,  278 

Tallquist,  T.  W.,  749 

Tammann,  G.,  enzymes,  12,  14 

Tangl,  Fr.,  blood-serum,  189,  190;  blood- 
analyses,  191,  236;  blood-sugar,  297; 
297;  development  of  the  egg,  511,  512, 
C  :  N  =  quotient,  628,  720 

Tanret,  Ch.,  524 

V.  Tappeiner,  H.,  celliilose,  397,  404;  bile- 
acids,  426 

V.  Tarchanoff,  J.,  bile  pigments,  331;  tata- 
proteid,  506 

Tarulli,  L.,  544 

Taveau,  R.,  278 

Tawara,  764 

Taylor,  A.  E.,  fatty  liver,  283;  fat  for- 
mation, 443;  lack  of  mineral  bodies,  735 

Tebb,  Chr.,  reticulin,  81;  glycogen,  289; 
amylolysis,  345,  388;  intestinal  inver- 
tase,  379;  cholesterin,  487 

Teeple,  J.,  bilirubin,  320,  321 

Teichmann,  L.,  htemin  crj'stals,  211,  649 

Tensstrom,  B.  St.,  bile,  311,  314 

Terfat,  P.,  616 

V.  Terray,  P.,  bile  and  putrefaction,  406, 
407;  oxalic  acid,  582;  lactic  acid  in  the 
urine,  608 

Terroine,  E.  F.,388 

Terry,  O.  P., 342 

Teruuchi,  Y.  ,  551 

Tetzner,  E.,  saliva,  343 


842 


INDEX  TO  AUTHORS. 


Thelen,  M.,  563 

Thesen,  J.,  isocreatinine,  455;  indican,  593 

Thiele,  O.,  613 

Thiemich,  M.,  283 

Thierfelder,  H.,  galactose,  120,  484; 
bariiini,  166;  digestion  and  micro- 
organisms, 405;  protagon,  482;  cere- 
bron  and  cerebrosides,  484,  485;  sphin- 
gosin,  4S5;    mammary  gland,  514,  539 

Thiroloix,  J.,  301 

Thiry,  L.,  intestinal  fistula  and  intestinal 
juice,  377,  378 

Thorner,  W.,  515 

Thompson,  W.  H.,  550 

Thorns,  H.,  556 

Thudichum,  L.  W.,  lecithins,  143;    bile- 
pigments,    321;      brain    phosphatides 
480-482,    485;     cerebrosides,    483-486 
luteins,      505;       urotheobromin,      580 
urinary  pigments,  601;    alcohol  in  the 
animal  body,  754 

Tiedemann,  F.,  347 

Tiemann,  H.,  milk,  522,  528 

Tigerstedt,  K.,  620 

Tigerstedt,  R.,  respiration  apparatus,  713, 
722;  metabolism,  729,  730,  756,  758, 
768 

Tissot,  J.,  468 

Tobler,  L.,  digestion,  370;  phosphaturia, 
621 

Toepfer,  G.,  617 

Tollens,  B.,  carbohydrates,  105,  111,  113, 
117,  118;  glucuronic  acid,  123;  urea,  556 

Tolmatscheff,  milk,  526,  531,  534 

Tompson,  F.  W.,  13 

Toppelius,  M.,  563 

Torup,  S.,  carbohsemoglobin,  206,  207; 
globulins,  701 

Tower,  81 

Toyonaga,  M.,  286 

Traube,  M.,  oxidations,  3,  6,  8 

Traube,  W.,  157 

Treupel,  G.,  urinary  carbohydrates,  609, 
659 

Trifanowski,  D.,  326 

Trillat,  A.,  19 

Tritschler,  F.,630 

Troller,  J.,  353 

Trommer,  C,  sugar  test,  116,  655 

Trumpy,  D.,  693 

Tschenloff,  B.,  744 

Tscherwinskv,  N.,  445 

Tschirjew,  S',  248 

Tsuboi,  J.,  244 

Tuczek.  F.,  346 

Tullner,  H.,  568 

Turby,  H.,  378 

V.  Udrdnsky,  L.,  diamines,  24,  615,  676; 
bile-acids,  312,  652;  urinary  pigments 
and  humin  substances,  601;  sugar  test, 
659;   cystine,  676 

TJffelmann,  J.,  374 

Uhlenhuth,  186 

Uhlik,  M.,  blood-crystals,  201,  203 


Ulpiani,  C,  lecithins,  146, 147;  brain,  480; 

uric  acid,  568 

Ulrich,  Chr.,  125 

Ultzmann,  R.,  680 

LTzer,  F.,  137 

Umber,  F.,  proteoses,  55;  nucleoproteids, 
150,  381;  transudates,  258;  gastric 
juice,  352,  353;    levulose,  665 

Umikoff,  N.,  532 

Underbill,  F.  P.,  234 

Urbain,  V.,  697 

Ure,  A.,  635 

Ussow,  393 

Ustjanzew,  W.,  427 

Vahlen,  E.,  bile-acids,  316,  317 
Valenciennes,  A.,  muscles  of  cephalopods, 

478;  yolk-spherules,  509 
Valenti,  A.,  532 
de  Vamossv,  Z.,  280 
Vandegrift'  G.  W.,  429 
Vandevelde,  A.  J.  J.,  523 
Vanlair,  C,  stercobilin,  320,  409 
Vaubel,  W.,  halogen  protein,  30;   Millon's 

reagent,  42;    estimation  of  acetone,  673 
Vauquelin,  L.  N.,  583 
\sLy,  Fr.,  liver  proteid,  282;  glycogen,  468 
V.  d.  Velde,  A.,  588 
V.  d.  Velden,  R.,  588 
Velichi,  J.,  477 
Vella,  L.,  377 
Veraguth,  O.,  744 

Verhaegen,  A.,  gastric  secretion,  353,  365 
Verneuil,  349 
Vernois,  M.,  534 
Vernon,  H.   M.,   erepsin,   379,   380,   391; 

pancreas  enzymes,  386,  388,  390,  391, 

396;   tryptic  digestion,  394 
Verploegh,  H.,  creatinine,  563,  567 
Verworm,  M.,  4 
Viault,  P.,  245 
Vierordt,    K.,    spectrophotometry,    218; 

expiration  air,  707 
Vigni,  L.,  413 
Vignon,  L.,  82 
Vila,  A.,  blood-  igments,  202,  205;    mus- 

culamine,  457 
Ville,  J.,  blood-catalases,  20;    blood-pig- 
ments, 202,  205,  212;    fat   absorption, 

425;  estimation  of  chlorine  in  urine,  616 
Villiers,  A.,  615 
Villiger,  V.,  6 
Vincent,  Sw.,  478 

Vines,  S.  H.,  plant-enzymes,  380,  390 
Viola,  E.,  191 
Virchow,   R.,   amyloid,   69;    hsematoidin 

216;    suprarenal  capsule,  330  » 

Vitali,  A.,  649 
Vitali,  D.,  616 
Vitek,  E.,  21 
Voltz,  W.,  milk-corpuscles,  517;  nutritive 

value  of  asparagin,  747 
Vogel.  J.,  pentoses,  110,  666;   amylysis, 

125,  344,  3S9;   lactase,  379 
Vogel,  R.,  467 


INDEX  TO   AUTHORS. 


843 


Vogelius,  288 

Voges.  O.,  548 

Vohl,  H.,  458 

Voirin.  G,,  597 

Voit.  C,  glycogen  formation,  291,  293. 
420;  bile  and  putrefact'on,  406;  fajces, 
408;  absorption.  413.  419,  424;  fa^ 
formation.  442-445,  742;  work  anJ 
metabolism,  471-473,  759;  nitrogen  in 
meat,  444,  476,  720;  urea  formation, 
553;  elimination  of  phosphoric  acid, 
620;  detection  of  milk  sugar,  665; 
st  ndard  figures  for  dietaries,  728,  750, 
764,  765,  767;  nitrogen  deficit,  718;  oxy- 
gen requirements,  723;  water  content  of 
the  body,  734;  starvation  metabolism, 
728,  729,  732,  738;  mineral  metabolism, 
735;  protein  catabolism,  740-744,  751; 
organized  and  circulating  protein,  742- 
744;  nutritive  value  of  gelatine,  745; 
proteoses,  746;  asparagin,  747;  water 
and  metabolism,  753;  salts,  753; 
dietaries,  764,  770.  771 

Voit,  E.,  f^t  determination,  137;  glycogen, 
293;  bones,  438,  439;  fat  formation, 
4-i4,  445;  heat  of  combustion,  727; 
starvation  metabolism,  730,  732,  738; 
vegetable  diets,  750;  law  of  surface,  757 

Voit,  Fr.,  fermentation  of  sugar,  120;  sugnr 
elimination,  293,  420;    thyroidea,  277; 

?;lycogen  formation,  293;    formation  of 
aeces,     409;      curare    poisoning,     467; 

acetone  bodies,  669,  670 
Volhard,    F.,    estimation  of  pepsin,  357; 

lipase,  363;    estimation  of  trypsin,  392 
Volhard,  J.,  titration  methods,  290,  581, 

617 
Volkmann,  A.  W.,  735 
van  Voornveld,  J.  A.,  246 
Vossius,  A.,  331 
Voswinckel,  H.,  690 
Vozarik,  A.,  acidity  of  urine,  544,  545 
Vulpian,  A.,  suprarenal  capsule,  277 

Wachsmann,  M.,  388 

Wachsmuth,  L.,  260 

Walchli,  G.,  76 

de  Waele,  H.,  523 

Wagner,  B.,  664 

Wagner,  H.,  extractives  of  the  mucle,  455. 

456 
Wahlgren,    V.,   biliary   mucus,   310,   329; 

glycocholeic  acid,  312 
Wait,  Ch.,  471 
Wakeman,  A.  J.,  284 
Waldvogel,    R.,   uric  acid,   571;    acetone 

bodies,  669 
Walter,  Fr.,  \irea  formation,  551;    blood- 

ga.ses,  701 
Walter,  G.,  ichthulin,  71,  504 
Walther,  A..  382 

V.  Walther,  P.,  fat  absorption,  421,  423 
Wanach,  R.,  220 
Wang,  E.,  594 
Wanklyn,  J.  A.,  518 


Wanner,   Fr.,  714 

Warren,  J.,  469 

Wasbutzki,  M.,  407 

Wassermann.  A.,  186 

Wassiliew,  W.,  387 

Wawrnsky,  R.,  368 

Waymouth  Reid  E.,  419 

Weber,  509 

Weber,  O.,  439 

Wedenski,  N.,  carbohydrate  of  the  urine, 
608 

Weidel,  H.,  xanthin  reaction,  160;  camine 
456 

Weigert,  Fr.,  98 

AVeigert,  R.,  185 

Weinland,  E.,  glycogen,  288,  293;  anti- 
enzymes,  355,  372,  380;  lactase,  379, 
386;   lactose  in  the  intestine,  420 

Weintraud,  W.,  diabetes,  300;  elimination 
of  nitrogen,  554;  phosphorus  meta- 
bolism, 619 

Weisbach,  486 

Weisgerber,  G.,  710 

Weiske,  H.,  cellulose  digestion,  397; 
bones,  438,  439;  nutritive  value  of  as- 
paragin,   747 

Weiss,  J.,  alkalinity  of  the  blood,  223; 
formation  of  sugar,  297;    uric  acid,  586 

Weiss,  H.  R.,  trypsin,  392,  393 

Weiss,  Sigm.,  glycogen,  291,  293,  468 

Weissberg,  J.,  6 

Wells,  H.  G.,  101 

V.  Wendt,  G.,  metabolism,  719,  738 

Wenz,  J.,  51 

Werenskiold,  F.,  529 

Werigo,  B.,  proteins,  38;    respiration,  710 

Wertheimer,  E.,  bile,  329;  pancreatic 
juice,  384 

Werther,  M.,  saliva,  347;  glycogen,  467; 
lactic  acid,  469 

Westphal,  C,  217 

Wetzel,  G.,  conchiolin,  81;    histidine,  99 

Weydemann,  H.,  32 

Weyl,  Th.,  protein  crystals,  38;  fibroin, 
82;  alanine,  84;  carbon  monoxide 
methsemoglobin,  207;  cholesterin,  334; 
muscle  work,  470;  amniotic  fluid,  512; 
creatinine,  565;  benzoic  acid,  588; 
nitrates,  623 

Wheeler,  H.  L.,  iodogorgonic  acid,  82; 
nucleic  acids,  156;  cytosine,  165 

White,  B.,  allantoin,  574,  584 

Wichmann,  A.,  proteins,  33,  181,  522 

Widdicombe,  J.  H.,  379 

Wiechowski,  W.,  hippuric  acid,  586,  587 

Wiener,  H.,  autolysis,  23;  uric  acid,  18, 
569,  571, 572,  573,  586;  oxalic  acid,  583 

Wild.  W.,  7 

Willdcnow,  C,  98 

Williams,  D.,  397 

Willstatter,  R.,  1  4 

Wiman.  A.,  47 

Windaus,  A.,  histidine.  99;  methylimi- 
dazole,  108;  cholesterin,  334 

Winkler,  441 


844 


INDEX   TO   AUTHORS. 


Winteler,  L.,  309 

Winter,    J.,    estimation    of    hydrochloric 

acid,  376;  milk,  530 
Winterberg,     H.,     ammonia,     241,     544; 

liver,  280;    carbamic-acid  intoxication, 

552 
Winternitz,    Hugo,    estimation   of    blood 

pigments,  217;  quantity  of  haemoglobin, 

243;    contents  of  the  gall-bladder,  329; 

putrefaction,  405,  588;  iodized  fat,  442, 

537,  538 
Wi  ternitz,    M.  C,  purine-base   enzymes, 

15S,  273,  571 
Winterstein,  E.,  amino-acids,  84,  87,  90; 

hexone  bases,  97,  98,  100;   inosite,  458; 

colostrum,    528;     tunicin,    686;     leci- 

thans,  144 
Wislicenus,  J.,  473 
Wittmdack,  K,  532 
Wohler,  Fr.,  synthesis  of  hippuric  acid, 

635;  ^of  urea,  547;  decomposition  of  uric 

acid,  573;  allantoin,  583 
Worner,  E.,  protagon,  482;  cerebron,  485; 

creatinine,  563;   uric  acid,  578 
Wohl,  A.,  106 
Wohlgemuth,   J.,   liver  proteid,   96,   282; 

oxyaminosuberic  acid,  96;   oxydiamino- 

sebacic  acid,   101;    pentoses,   109,   111, 

112,  290;   glycogen  formation,  290,  292; 

sulphurized    products    of    metabolism, 

331;     enzymes    of    the   egg-yolk,    503; 

amino-acids,  demolition  of,  636 
Wolff,  E.,  471 
Wolff,  H.,  glucosamine,  121;  transudates, 

262;  melanins,  689 
Wolff,  L.  K.,  46 
Wolff  berg,   S.,  glycogen  formation,  293; 

respiration,  708,  709 
Wolkow,  M.,  597 
Woll,  F.  W.,  516 
Woltering,    H.,    iron    preparations,    245 

liver-proteid,  282 
Woods,  H.  S.,  487 
Wooldridge,  L.  C,  tissue-fib rinogen,  141 

stroma  of  blood-corpuscles,  194;  coagvi- 

lation  of  the  blood,  228,  233,  234 
Worm-Muller;  J.    ,blood,  244,  245,  248 

sugar  test,  655;    estimation  of  sugar, 

660,  662,  663 
Wright,    A.,    fibrin   ferment    and    coagu- 
lation, 175,  226,  233;    alkalinity  of  the 

blood,  223;   phlorhizin  diabetes,  298 
Wroblewsky,      A.,      fermentation,      10; 

pseudonuclein,  47;  starch,  126;  pepsin, 

354,  358;    gastric  digestion,  370,  371; 

action  of  enzymes,  397;   milk,  531 
Wulff,  C,  purine  bases,  161,  164,  580 
Wurm,  W.  A.,  690 
Wurster,  C,  625 
Wurtz,  A.,  lymph,  251;   colloid,  499 

Young,  P.  A.,  325 

Young,    R.    A.,    dextrin,   129;    glycogen, 

289 
Yvon,  P.,  611 


Zadik,  H.,  719 

Zangerle,  M.,  500 

Zahor,  H.,  646 

Zaitschek,    A.,    enzymes    of    milk,    523; 

casein,  521,  529,  531 
Zak,  E.,  258 
Zaleski,    J.,   plant-   and   blood-pigments, 

197,  214;  blood-pigments,  211,  2^3,  214j 

216;     hsemopyrrol,    214;     ammonia   in 

the  blood,  241,  551;    in  the  glands,  624, 

774;    estimation  of,  625;    formation  of 

urea,  553,  554;   estimation  of  urea,  561; 

liver  and  acid  formation,  554,  572 
Zaleski,  St.,  iron  of  liver,  282,  286:    iron 

in    sucklings,     535;     reaction    of    the 

intestine,  407;    milk,  536 
Zalesky,     N.,     bones,    436;     samandarin, 

692 
Zalocostas,  P.,  81 
Zander,  E.,  686 
Zanetti,   C.   U.,   glucoproteid,    180;    bile 

310;   ovomucoid,  508 
Zangemeister,    W.,    estimation   of   blood- 
pigments,  217;   amniotic  fluid,  513 
Zaudy,  569 
Zdarek,  E.,  688 
v.  Zebrowski,  E.,  342 
Zeehuizen,  H.,  631 
Zeidlitz,  P.  V.,  656 
Zsitler,  X.,  470 
Zeller,  A.,   elimination  of  chlorine,   616; 

destruction    of     cWorine     compounds, 

631 
V.  Zeynek,  R.,  fat  of  dermoid  cysts,  138, 

502,    691;     blood-pigments,    204,    205, 

209;    liver,  286;    bile,  327;    sarcomela- 

nin,  689 
Zickgraf,   G.,   oxidation   of  proteins,    32, 

78 
Ziegler,  583 
Ziegler,  E.,  635 
Zillesen,  H.,  469 
de  Zilva,  L.,  387 
Zimnitzki,  S.,  405 
Zink,  J.,  fats,  135,  437,  442 
Zinoffsky,  O.,  197 
Zinsser,  A.,  363 
Zobel,  S.,  459 
Zoja,  L.,  oxyproteic  acids,  31;  elastin,  75, 

76;     ovalbumin,    507:      urobilin,    604; 

uroerythyrin,    607;     hsematoporphyrin, 

649,  650 
Zsigmondy,  R.,  41 
Zuelzer,   G.,   lecithin,    147;     myelin,   486; 

cutaneous  respiration,  695 
V.  Zumbusch,  L.,  324 
Zunz,    E.,    digestion    jDroducts,    53,    55; 

digestion,  370,  418;    trypsinogen,  386; 

absorption,  413;    extractives  of  meat, 

457 
Zuntz,  L.,  223 
Zuntz,  N.,  blood,  224,  243,  246;  glycogen, 

288;   blood-sugar.  296.  469;    phlorhizin 

diabetes,    298:     digestion,    393,     427; 

protein  assimilation,   412;    muscle- fat. 


INDEX  TO   AUTHORS. 


845 


463  472;  muscle  metabolism,  467,  471, 
473-  high  altitudes,  246,  694,  761; 
cutaneous  respiration,  695;  blood-gases, 
697-699;  alveolar  air,  707:  respiration, 
710  712  713,  722,  723,  733;  metabo- 
lism, 723,  729,  755,  757,  758,  761 ;  nutri- 


tive  value  of  proteoses,  746;  of  alcohol, 
754;    digestibility  of  bread,  762 

Zweifel,  P.,  ptyalin,  344;  pancreatic 
diastase.  388;    eclampsia,  608 

Zwerger,  R.,  58,  59 


